Method for reducing immunogenicity of rna

ABSTRACT

The present invention relates to RNA therapy and, in particular, decreasing immunogenicity of RNA. Specifically, the present invention provides methods for decreasing immunogenicity of RNA, said methods comprising modifying the nucleotide sequence of the RNA by reducing the uridine (U) content, wherein said reduction of the U content comprises an elimination of U nucleosides from the nucleotide sequence of the RNA and/or a substitution of U nucleosides by nucleosides other than U in the nucleotide sequence of the RNA. Using RNA having decreased immunogenicity allows administration of RNA as a drug to a subject, e.g. in order to obtain expression of a pharmaceutically active peptide or protein, without eliciting an immune response which would interfere with therapeutic effectiveness of the RNA or induce adverse effects in the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. Ser. No. 15/755,309, filed onFeb. 26, 2018, which is a U.S. National Stage of PCT/EP2016/070012,filed on Aug. 24, 2016, which is a continuation-in-part of InternationalApplication No. PCT/EP2015/069760, filed on Aug. 28, 2015, each of whichis incorporated herein by reference.

SEQUENCE LISTING INCORPORATION

Biological sequence information for this application is included in anASCII text file, having the file name “ZSP-136-SEQ-V2.txt” created onJul. 24, 2018, and a having file size of 14,819 bytes, which isincorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to RNA therapy and, in particular,decreasing immunogenicity of RNA. Specifically, the present inventionprovides methods for decreasing immunogenicity of RNA, said methodscomprising modifying the nucleotide sequence of the RNA by reducing theuridine (U) content, wherein said reduction of the U content comprisesan elimination of U nucleosides from the nucleotide sequence of the RNAand/or a substitution of U nucleosides by nucleosides other than U inthe nucleotide sequence of the RNA. Using RNA having decreasedimmunogenicity allows administration of RNA as a drug to a subject, e.g.in order to obtain expression of a pharmaceutically active peptide orprotein, without eliciting an immune response which would interfere withtherapeutic effectiveness of the RNA or induce adverse effects in thesubject.

BACKGROUND OF THE INVENTION

In vitro-transcribed mRNA (IVT mRNA) is emerging as a new drug classthat has the potential to play an important role in gene therapy. Whilefirst described as a therapeutic in 1992, the immunogenicity of IVT mRNAprevented its development for protein replacement therapies. However,this problem was solved by introducing modified nucleosides into mRNA(see Karikó, K., Buckstein, M., Ni, H., and Weissman, D. (2005)Suppression of RNA recognition by Toll-like receptors: the impact ofnucleoside modification and the evolutionary origin of RNA. Immunity 23,165-175). In this study, all uridines were exchanged for pseudouridines,the most common naturally occurring modified nucleoside.Pseudouridine-modified mRNA was found to be highly translatable andnon-immunogenic (see Karikó, K., Muramatsu, H., Welsh, F. A., Ludwig,J., Kato, H., Akira, S., and Weissman, D. (2008) Incorporation ofpseudouridine into mRNA yields superior nonimmunogenic vector withincreased translational capacity and biological stability. Moleculartherapy 16, 1833-1840). An alternative solution was to replace 25% ofthe uridine residues with 2-thiouridine (s2U), resulting in an mRNA withsome residual immunogenicity (see Kormann, M. S., Hasenpusch, G., Aneja,M. K., Nica, G., Flemmer, A. W., Herber-Jonat, S., Huppmann, M., Mays,L. E., Illenyi, M., Schams, A., Griese, M., Bittmann, I., Handgretinger,R., Hartl, D., Rosenecker, J., and Rudolph, C. (2011) Expression oftherapeutic proteins after delivery of chemically modified mRNA in mice.Nature biotechnology 29, 154-157).

Recently, an alternative method for generating therapeuticallyapplicable IVT mRNA was reported that does not require the use ofmodified nucleosides (see Thess, A., Grund, S., Mui, B. L., Hope, M. J.,Baumhof, P., Fotin-Mleczek, M., and Schlake, T. (2015)Sequence-engineered mRNA without chemical nucleoside modificationsenables an effective protein therapy in large animals. Molecular Therapy23, 1457-1465). In this study, the sequence composition of the mRNA wasaltered by selecting codons with the highest GC-rich content for eachamino acid. The study indicates that such GC-maximized mRNAs may havethe potential to reduce immune activation and thereby to improvetranslation and half-life of the mRNA.

However, lack of immunogenicity of the codon-optimized GC-maximizedsequences has not been demonstrated unequivocally in this study sincethe test IVT mRNA was formulated with TransIT®, a commercially availablecomplexing agent. It is known that TransIT®-formulated RNA primarilyinduces IFN-α (see Karikó, K., Muramatsu, H., Ludwig, J., and Weissman,D. (2011) Generating the optimal mRNA for therapy: HPLC purificationeliminates immune activation and improves translation ofnucleoside-modified, protein-encoding mRNA. Nucleic acids research 39,e142), but IFN-α induction was not measured in any of the experimentsperformed by Thess and colleagues. As a consequence, the level ofreduction of immune activation by means of using GC-maximized mRNAs andthe therapeutical benefit of this method remains unclear.

Thus, it is an object of the present invention to provide an alternativeand even superior method for generating therapeutically applicable andnon-immunogenic RNA, in particular IVT mRNA which likewise does notrequire the use of modified nucleosides.

It is demonstrated herein that mRNA constructs with low uridine andincreased adenosine content have low immunogenicity. Both immunogenicityand translatability of these A-rich (U-poor) mRNAs were compared to thecorresponding wild-type (wt) mRNAs in vitro in human dendritic cells(DCs) and in vivo in BALB/c mice.

SUMMARY OF THE INVENTION

The present invention is directed to methods of altering RNA such aseukaryotic, preferably mammalian, mRNA which result in a reducedimmunogenicity of the RNA and enables its use in RNA therapy, e.g. toprovide a peptide or protein of interest. The invention also pertains tocompositions comprising such RNA. The invention also relates to RNAtherapy, e.g., the use of RNA described herein as a drug in order toobtain the expression of a therapeutically relevant peptide or proteinwithin a cell. The subject compositions and methods are useful intreating a myriad of disorders involving errors in expression ofproteins.

In one aspect the invention relates to a method of decreasingimmunogenicity of RNA, said method comprising modifying the nucleotidesequence of the RNA by reducing the uridine (U) content, wherein saidreduction of the U content comprises an elimination of U nucleosidesfrom the nucleotide sequence of the RNA and/or a substitution of Unucleosides by nucleosides other than U in the nucleotide sequence ofthe RNA.

In one embodiment, the method comprises the steps of:

(i) providing the nucleotide sequence of a first RNA,

(ii) designing the nucleotide sequence of a second RNA, said nucleotidesequence of the second RNA comprising a reduced U content compared tothe nucleotide sequence of the first RNA, and, optionally,

(iii) providing the second RNA.

In a further aspect the invention relates to a method of providing anucleic acid molecule for RNA transcription comprising the steps of:

(i) providing a first DNA sequence encoding the nucleotide sequence of afirst RNA,

(ii) designing a second DNA sequence encoding the nucleotide sequence ofa second RNA, said nucleotide sequence of the second RNA comprising areduced U content compared to the nucleotide sequence of the first RNA,wherein said reduction of the U content comprises an elimination of Unucleosides from the nucleotide sequence of the RNA and/or asubstitution of U nucleosides by nucleosides other than U in thenucleotide sequence of the RNA, and

(iii) providing a nucleic acid molecule comprising the second DNAsequence.

In one embodiment of the methods of the invention, the RNA encodes atleast one peptide or protein. In one embodiment, the peptide or proteinis pharmaceutically active or antigenic. In one embodiment, the aminoacid sequence of the peptide or protein encoded by the RNA modified byreducing the U content is identical to the amino acid sequence of thepeptide or protein encoded by the non-modified RNA.

In one embodiment of the methods of the invention, said reduced Ucontent renders the RNA modified by reducing the U content lessimmunogenic compared to the non-modified RNA.

In one embodiment of the methods of the invention, the U content in theRNA modified by reducing the U content is reduced by at least 10%,preferably at least 20%, more preferably at least 30% compared to thenon-modified RNA.

In one embodiment of the methods of the invention, the U content isreduced in one or more of the 5′ untranslated region, the coding regionand the 3′ untranslated region of the RNA. In one embodiment of themethods of the invention, the U content is reduced in the coding regionof the RNA.

In one embodiment of the methods of the invention, said reduction of theU content comprises a substitution of U nucleosides by nucleosides otherthan U in the nucleotide sequence of the RNA.

In one embodiment of the methods of the invention, said nucleosidesother than U are selected from the group consisting of adenosine (A),guanosine (G), 5-methyluridine (m5U) and cytidine (C).

In one embodiment of the methods of the invention, said reduction of theU content comprises a substitution of U nucleosides by adenosine (A)nucleosides.

In one embodiment of the methods of the invention, said reduction of theU content comprises altering codons which comprise at least one Unucleoside by other codons that encode the same amino acids but comprisefewer U nucleosides and preferably comprise no U nucleosides.

In one embodiment, the methods of the invention further compriseintroducing at least one analogue of a naturally occurring nucleosideinto the nucleotide sequence of the RNA. In one embodiment, introducingthe analogue of a naturally occurring nucleoside into the nucleotidesequence of the RNA reduces immunogenicity of the RNA. In oneembodiment, introducing at least one analogue of a naturally occurringnucleoside into the nucleotide sequence of the RNA comprises asubstitution of U nucleosides by pseudouridines.

In one embodiment of the methods of the invention, the RNA is mRNA.

In a further aspect the invention relates to a method of obtaining RNAcomprising the steps of (i) providing a nucleic acid molecule for RNAtranscription according to the method of the invention of providing anucleic acid molecule for RNA transcription, and (ii) transcribing RNAusing the nucleic acid molecule as a template.

In a further aspect the invention relates to a modified RNA havingdecreased immunogenicity compared to naturally occurring RNA, saidmodified RNA having a nucleotide sequence comprising a reduced U contentcompared to said naturally occurring RNA, wherein said reduction of theU content comprises an elimination of U nucleosides from the nucleotidesequence of the RNA and/or a substitution of U nucleosides bynucleosides other than U in the nucleotide sequence of the RNA.

Embodiments of the RNA of the invention are as described above for themethods of the invention.

RNA described herein may be employed, for example, for transientexpression of genes, with possible fields of application being RNApharmaceuticals which are administered for transient expression offunctional recombinant proteins such as erythropoietin, hormones,coagulation inhibitors, etc., in vivo.

In a further aspect the invention relates to a method of treating asubject using RNA comprising the steps of (i) decreasing immunogenicityof RNA according to the method of the invention of decreasingimmunogenicity of RNA, and (ii) administering the RNA to the subject.

In a further aspect the invention relates to a method of treating asubject using RNA comprising the steps of (i) obtaining RNA according tothe method of the invention of obtaining RNA, and (ii) administering theRNA to the subject.

In a further aspect the invention relates to a method of treating asubject comprising administering the RNA of the invention to thesubject.

In one embodiment, the RNA described herein is administered repetitivelyto a subject. In one embodiment, the RNA described herein isadministered to a subject so as to be introduced into cells of thesubject for expression of the peptide or protein encoded by the RNA.

In one particularly preferred embodiment, the RNA described herein is invitro transcribed RNA. In one embodiment, the RNA described herein ismodified by pseudouridine and/or 5-methylcytidine.

In one embodiment, decreasing the U content results in a reduction ofimmunogenicity of the RNA compared to the situation where the U contentis not reduced.

In one particularly preferred embodiment of the invention, the RNA whichis modified by reducing the U content has an increased A content andpreferably a reduced GC content compared to non-modified RNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows (A) the nucleotide compositions of mRNAs coding for wildtype murine EPO (wt EPO), GC-rich murine EPO (also called optimizedmurine EPO/omEPO), A-rich murine EPO, and GC-maximized murine EPO asdescribed in Thess, A., Grund, S., Mui, B. L., Hope, M. J., Baumhof, P.,Fotin-Mleczek, M., and Schlake, T. (2015); and (B) shows the nucleotidecompositions of mRNAs coding for wild type canine EPO (wt EPO) andA-rich canine EPO (cEPO).

FIG. 2 shows levels of EPO & IFN alpha (IFN-α) in plasma at 6/24 hfollowing injection of 10 μg LNP-formulated murine EPO mRNA, which wereprepared according to example 2. The lowest level of IFN-α was inducedby the A-rich mRNA that contained the lowest number of uridines. TheA-rich RNA translated more efficiently and 5-times more EPO was producedfrom it compared to the wt EPO RNA. These results demonstrate directcorrelation between the U-content of the RNA and its immunogenicity, asthe wt RNA with the highest U content induced the most IFN-α, while theA-rich mRNA that contained the lowest number of uridine induced theleast IFN-α.

FIG. 3 shows levels of EPO & IFN alpha in plasma at 6/24 h followinginjection of 20 μg mRNA-liposomal formulation which were preparedaccording to example 4. The results were similar to those obtained withLNP-formulated mRNA since the wt RNA with the highest U content inducedthe most IFN-α, while the A-rich mRNA induced the least IFN-α.

FIG. 4 shows IFN alpha induction and EPO production by human DCstransfected with 0.1 μg TransIT-complexed mouse EPO mRNA. Interferonalpha (IFN-α) and murine EPO levels were measured in the culture mediumof human monocyte-derived dendritic cells at 24 h following exposure toTransIT-complexed 0.1 μg EPO mRNAs. A-rich EPO mRNA, which contained theleast uridine (U) in their coding sequences (CDS) secreted the most EPOprotein and induced significantly less IFN-α than the GC-rich or the wtEPO mRNAs.

FIG. 5 shows a comparison of the uridine contents of murine EPO encodingwild type mRNA, murine EPO encoding GC-rich mRNA and murine EPO encodingA-rich mRNA.

FIG. 6 shows hematocrit values obtained in mice (n=5) before (Day 0) andat day 7 and 14 after intraperitoneal injection of 3 μg ofTransIT-complexed canine EPO mRNA. By day 7 hematocrits increasedsignificantly in all mice injected with A-rich canine EPO mRNA, whichcontained the least uridine (U) and had the lowest GC (Guanosine &Cytidine) content in their coding sequences (CDS). At day 7 followingadministration of A-rich canine EPO mRNA, the hematocrits weresignificantly higher than in mice injected with the wt canine EPOmRNA. * Denotes p value <0.05. Hematocrits were measured by drawing lessthan 20 μL of blood as described by Mahiny and Kariko (Methods Mol Biol1428: 297-306, 2016), thus avoiding blood loss-related hematocritincrease.

FIG. 7 shows a comparison of the uridine contents of coding sequencesfor canine EPO-encoding wild type mRNA and canine EPO-encoding A-richmRNA.

DETAILED DESCRIPTION OF THE INVENTION

Although the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kölbl, Eds.,Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, andrecombinant DNA techniques which are explained in the literature in thefield (cf., e.g., Molecular Cloning: A Laboratory Manual, 2^(nd)Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press,Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step or group of members, integers orsteps but not the exclusion of any other member, integer or step orgroup of members, integers or steps although in some embodiments suchother member, integer or step or group of members, integers or steps maybe excluded, i.e. the subject-matter consists in the inclusion of astated member, integer or step or group of members, integers or steps.The terms “a” and “an” and “the” and similar reference used in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”), provided herein isintended merely to better illustrate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

The term “immunogenicity” refers to the ability of a particularsubstance, in particular RNA, to provoke an immune response in the bodyof an animal such as a human. In other words, immunogenicity is theability to induce a humoral and/or cell mediated immune response.Unwanted immunogenicity includes an immune response by an organismagainst a therapeutic substance such as a drug. This reaction mayinactivate the therapeutic effects of the treatment and may induceadverse effects.

The RNA described herein which is modified by reducing the U content issignificantly less immunogenic than an unmodified RNA moleculecontaining more U. In one embodiment, the modified RNA is at least 5%less immunogenic than its unmodified counterpart. In another embodiment,immunogenicity is reduced by at least 10%. In another embodiment,immunogenicity is reduced by at least 20%. In another embodiment,immunogenicity is reduced by at least 30%. In another embodiment,immunogenicity is reduced by at least 40%. In another embodiment,immunogenicity is reduced by at least 50%. In another embodiment,immunogenicity is reduced by at least 60%. In another embodiment,immunogenicity is reduced by at least 70%. In another embodiment,immunogenicity is reduced by at least 80%. In another embodiment,immunogenicity is reduced by at least 90%. In another embodiment,immunogenicity is removed or essentially removed, i.e. reduced by about100%. The relative immunogenicity of the modified RNA and its unmodifiedcounterpart may be determined by determining the quantity of theunmodified RNA required to elicit the same result to the same degree(e.g. expression of the same amount of protein) as a given quantity ofthe modified RNA. For example, if twice as much unmodified RNA isrequired to elicit the same response, then the modified RNA is 50% lessimmunogenic than the unmodified RNA. In another embodiment, the relativeimmunogenicity of the modified RNA and its unmodified counterpart isdetermined by determining the quantity of cytokine (e.g. IL-12, IFN-α,TNF-α, RANTES, MIP-1α or β, IL-6, IFN-β, or IL-8) secreted in responseto administration of the modified RNA, relative to the same quantity ofthe unmodified RNA. For example, if one-half as much cytokine issecreted, then the modified RNA is 50% less immunogenic than theunmodified RNA.

“Significantly less immunogenic” refers to a detectable decrease inimmunogenicity. In another embodiment, the term refers to a decreasesuch that an effective amount of the RNA can be administered orrepeatedly administered without triggering a detectable immune response.In another embodiment, the term refers to a decrease such that the RNAcan be repeatedly administered without eliciting an immune responsesufficient to detectably reduce expression of the peptide or proteinencoded by the RNA. In another embodiment, the decrease is such that theRNA can be repeatedly administered without eliciting an immune responsesufficient to eliminate expression of the peptide or protein encoded bythe RNA.

Terms such as “decreasing”, “reducing” or “inhibiting” relate to theability to cause an overall decrease, preferably of 5% or greater, 10%or greater, 20% or greater, more preferably of 50% or greater, and mostpreferably of 75% or greater, in the level. This also includes acomplete or essentially complete decrease, i.e. a decrease to zero oressentially to zero.

Terms such as “increasing”, “enhancing”, or “prolonging” preferablyrelate to an increase, enhancement, or prolongation by about at least10%, preferably at least 20%, preferably at least 30%, preferably atleast 40%, preferably at least 50%, preferably at least 80%, preferablyat least 100%, preferably at least 200% and in particular at least 300%.These terms may also relate to an increase, enhancement, or prolongationfrom zero or a non-measurable or non-detectable level to a level of morethan zero or a level which is measurable or detectable.

As demonstrated herein, immunogenicity of RNA can be decreased byreducing the U content of the RNA, i.e. reducing the percentage of Unucleosides in the RNA. Reducing the U content of the RNA can beaccomplished by eliminating U nucleosides from the nucleotide sequenceof the RNA and/or by substituting U nucleosides by nucleosides otherthan U in the nucleotide sequence of the RNA.

“Eliminating U nucleosides from the nucleotide sequence of the RNA”means that U nucleosides are deleted from an RNA sequence. In oneembodiment, U nucleosides are eliminated from the non-coding regions ofan mRNA molecule. In one embodiment, the U nucleosides are eliminatedfrom the 5′ untranslated region (UTR) and/or the 3′ UTR of an mRNAmolecule. In one embodiment of the invention, at least 5%, at least 10%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, orat least 70% of the U nucleosides are eliminated.

“Substituting U nucleosides by nucleosides other than U in thenucleotide sequence of the RNA” means that U nucleosides are deletedfrom an RNA sequence and the same number of nucleosides other than U areinserted, e.g. in place of the deleted U nucleosides. Thus,“substituting U nucleosides by nucleosides other than U in thenucleotide sequence of the RNA” means that the U content is reducedwithout removing nucleotides and thus, reducing the number ofnucleotides in the RNA. U nucleosides may be substituted in thenon-coding regions and/or coding regions of an mRNA molecule. In oneembodiment, U nucleosides are substituted in the coding regions of anmRNA molecule. In one embodiment, the U content is reduced bysubstituting one codon encoding a particular amino acid by another codonencoding the same or a related amino acid, preferably the same aminoacid, and containing less U. The degeneracy of the genetic code willallow the number of U nucleosides that are present in the non-modifiedsequence to be reduced, while maintaining the same coding capacity.

It is preferred according to the invention that the nucleotide sequenceof the RNA is modified by reducing the U content in the coding region ofthe RNA by substituting U containing codons by other codons encoding thesame amino acids but comprising fewer and preferably no U nucleosides sothat the amino acid sequence of the peptide or protein encoded by themodified RNA is identical to the amino acid sequence of the peptide orprotein encoded by the non-modified RNA. In one particularly preferredembodiment, the U content is reduced to the highest extent possible.

Depending on which amino acid is encoded by a codon, several differentpossibilities for modification of RNA sequences may be possible. In thecase of amino acids encoded by codons that comprise exclusively A, C orG, no modification would be necessary to reduce the U content. In othercases, codons which comprise U nucleosides can be altered by simplysubstituting other codons that encode the same amino acids but that donot comprise U nucleosides or comprise fewer U nucleosides. For example:

the codons for Arg can be altered to AGA, AGG, CGC, CGA or CGG,preferably AGA instead of CGU;the codons for Gly can be altered to GGC, GGA or GGG, preferably GGAinstead of GGU;the codons for Pro can be altered to CCC, CCA or CCG, preferably CCAinstead of CCU;the codons for Thr can be altered to ACC, ACA or ACG, preferably ACAinstead of ACU;the codons for Ala can be altered to GCC, GCA or GCG, preferably GCAinstead of GCU;the codons for Leu can be altered to CUC, CUA or CUG, preferably CUG orCUC instead of UUA, UUG or CUU;the codons for Ile can be altered to AUC or AUA, preferably AUC insteadof AUU;the codons for Val can be altered to GUC, GUA or GUG, preferably GUGinstead of GUU;the codons for Ser can be altered to UCC, UCA, UCG or AGU instead ofUCU;preferably:the codons for Ser can be altered to AGC instead of UCU, UCC, UCA, UCGor AGU;the codons for Phe can be altered to UUC instead of UUU;the codons for Asn can be altered to AAC instead of AAU;the codons for His can be altered to CAC instead of CAU;the codons for Tyr can be altered to UAC instead of UAU;the codons for Asp can be altered to GAC instead of GAU;the codons for Cys can be altered to UGC instead of UGU.

However, there are instances in which the U content of particular codonscannot be altered by sequence changes and still encode the same aminoacid. For instance:

Met—AUG; Stop—UAA, UAG or UGA; Trp—UGG.

In a one embodiment of the invention, at least 5%, at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% or 100% of the codons whichcomprise U nucleosides and can be substituted by other codons thatencode the same amino acids but that do not comprise U nucleosides orcomprise fewer U nucleosides are substituted by other codons that encodethe same amino acids but that do not comprise U nucleosides or comprisefewer U nucleosides. In one particularly preferred embodiment, at leastas many of the codons which comprise U nucleosides and can besubstituted by other codons that encode the same amino acids but that donot comprise U nucleosides or comprise fewer U nucleosides aresubstituted by other codons that encode the same amino acids but that donot comprise U nucleosides or comprise fewer U nucleosides such thatimmunogenicity of the RNA is decreased.

In one embodiment of the invention, at least 5%, at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least70% of the U nucleosides are eliminated by substitution. It is preferredaccording to the invention that the U content of the RNA is reduced byat least 5%, preferably at least 10%, preferably at least 15%,preferably at least 20%, preferably at least 30% or preferably at least40% and up to 80%, preferably up to 70%, preferably up to 60%, orpreferably up to 50%. If the nucleotide sequence of the RNA is onlymodified by reducing the U content in the coding region of the RNA bysubstituting U containing codons by other codons encoding the same aminoacids but comprising fewer and preferably no U nucleosides the above mayrelate to the nucleotide sequence of the coding region only.

It is preferred according to the invention that in the modified RNAhaving a reduced U content, the GC content is not significantlyincreased compared to the non-modified RNA. In this respect, “notsignificantly increased” means that the GC content is increased by atmost 10%, preferably at most 5%, more preferably at most 3%, 2% or 1%.In one particularly preferred embodiment, the GC content is notincreased, i.e. it remains essentially constant, or is reduced. In thisrespect, “reduced GC content” preferably means that the GC content isreduced by at least 1%, preferably at least 2%, preferably at least 3%,more preferably at least 4%, at least 5%, at least 6%, at least 7%, atleast 8%, at least 9% or at least 10%. If the nucleotide sequence of theRNA is only modified by reducing the U content in the coding region ofthe RNA by substituting U containing codons by other codons encoding thesame amino acids but comprising fewer and preferably no U nucleosidesthe above may relate to the nucleotide sequence of the coding regiononly.

It is preferred according to the invention that in the modified RNAhaving a reduced U content, the A content is increased compared to thenon-modified RNA. In this respect, “increased A content” preferablymeans that the A content is increased by at least 1%, preferably atleast 3%, preferably at least 5%, preferably at least 10%, morepreferably at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 37%, or at least 40%. If the nucleotide sequence ofthe RNA is only modified by reducing the U content in the coding regionof the RNA by substituting U containing codons by other codons encodingthe same amino acids but comprising fewer and preferably no Unucleosides the above may relate to the nucleotide sequence of thecoding region only.

An increase in A content (and non significant increase in GC content,preferably constant GC content or reduction in GC content) can beachieved by altering codons which comprise U nucleosides bysubstituting—at least partially—other codons that encode the same aminoacids but that do not comprise U nucleosides or comprise fewer Unucleosides and have a high A content and a low GC content. For example:

the codons for Arg can be altered to AGA instead of CGU;the codons for Gly can be altered to GGA instead of GGU;the codons for Pro can be altered to CCA instead of CCU;the codons for Thr can be altered to ACA instead of ACU;the codons for Ala can be altered to GCA instead of GCU.

Alternatively or additionally, an increase in A content (and nonsignificant increase in GC content, preferably constant GC content orreduction in GC content) can be achieved by altering codons bysubstituting—at least partially—other codons that encode the same aminoacids, do not comprise more U nucleosides—preferably no U nucleosides asthe substituted codons—and have a high A content and a low GC content.For example:

the codons for Arg can be altered to AGA instead of CGG, AGG, CGC andCGA;the codons for Gly can be altered to GGA instead of GGC and GGG;the codons for Pro can be altered to CCA instead of CCC and CCG;the codons for Thr can be altered to ACA instead of ACC and ACG;the codons for Ala can be altered to GCA instead of GCC and GCG;the codons for Glu can be altered to GAA instead of GAG;the codons for Lys can be altered to AAA instead of AAG.

In one particularly preferred embodiment, the modified RNA having areduced U content is A-rich (or A-enriched compared to the non-modifiedRNA, i.e. its A content is increased).

The term “A-rich” as used herein refers to nucleic acid molecules withan A content of more than 25%. In particular aspects, the A-rich nucleicacid comprises about 30% A to about 37% A, and in additional aspects,the A-rich nucleic acid comprises greater than about 26% A, greater thanabout 27% A, greater than about 28% A, greater than about 29% A, greaterthan about 30% A, greater than about 31% A, greater than about 32% A,greater than about 33% A, greater than about 34% A, greater than about35% A, greater than about 36% A, and so forth. If the nucleotidesequence of the RNA is only modified by reducing the U content in thecoding region of the RNA by substituting U containing codons by othercodons encoding the same amino acids but comprising fewer and preferablyno U nucleosides the above may relate to the nucleotide sequence of thecoding region only.

In one embodiment, an A-rich RNA according to the invention has a GCcontent of less than about 60%. In another embodiment, an A-rich RNA hasa GC content of less than about 55%. In another embodiment, an A-richRNA has a GC content of less than about 54%. In another embodiment, anA-rich RNA has a GC content of less than about 53%. In anotherembodiment, an A-rich RNA has a GC content of less than about 52%. Inanother embodiment, an A-rich RNA has a GC content of less than about51%. In another embodiment, an A-rich RNA has a GC content of less thanabout 50%. If the nucleotide sequence of the RNA is only modified byreducing the U content in the coding region of the RNA by substituting Ucontaining codons by other codons encoding the same amino acids butcomprising fewer and preferably no U nucleosides the above may relate tothe nucleotide sequence of the coding region only.

The term “GC-rich” as used herein refers to nucleic molecules with a G+Ccontent of more than 50%. In particular aspects, the GC-rich nucleicacid comprises about 60% GC to about 75% GC, and in additional aspects,the GC-rich nucleic acid comprises greater than about 55% GC, greaterthan about 60% GC, greater than about 61% GC, greater than about 62% GC,greater than about 63% GC, greater than about 64% GC, greater than about65% GC, greater than about 66% GC, greater than about 67% GC, greaterthan about 68% GC, greater than about 69% GC, greater than about 70% GC,and so forth. If the nucleotide sequence of the RNA is only modified byreducing the U content in the coding region of the RNA by substituting Ucontaining codons by other codons encoding the same amino acids butcomprising fewer and preferably no U nucleosides the above may relate tothe nucleotide sequence of the coding region only.

In one embodiment, GC-rich RNA according to the invention has an Acontent of less than about 30%. In another embodiment, a GC-rich RNA hasan A content of less than about 25%. In another embodiment, a GC-richRNA has an A content of less than about 24%. In another embodiment, aGC-rich RNA has an A content of less than about 23%. In anotherembodiment, a GC-rich RNA has an A content of less than about 22%. Inanother embodiment, a GC-rich RNA has an A content of less than about21%. In another embodiment, a GC-rich RNA has an A content of less thanabout 20%. If the nucleotide sequence of the RNA is only modified byreducing the U content in the coding region of the RNA by substituting Ucontaining codons by other codons encoding the same amino acids butcomprising fewer and preferably no U nucleosides the above may relate tothe nucleotide sequence of the coding region only.

As used herein, the term “U content” refers to the amount of nucleosidesof a particular RNA molecule or RNA sequence that are uridine (U)typically expressed as a percent. Where the sequence of particular RNAis known, the U content can be determined using the formula:

$\frac{U}{A + U + G + C} \times 100$

wherein G, C, A and U refer to the number of each residue in theparticular RNA molecule or RNA sequence, to provide a percent U content.If the nucleotide sequence of the RNA is only modified by reducing the Ucontent in the coding region of the RNA by substituting U containingcodons by other codons encoding the same amino acids but comprisingfewer and preferably no U nucleosides the above may relate to thenucleotide sequence of the coding region only.

As used herein, the term “A content” refers to the amount of nucleosidesof a particular RNA molecule or RNA sequence that are adenosine (A)typically expressed as a percent. Where the sequence of particular RNAis known, the A content can be determined using the formula:

$\frac{A}{A + U + G + C} \times 100$

wherein G, C, A and U refer to the number of each residue in theparticular RNA molecule or RNA sequence, to provide a percent A content.If the nucleotide sequence of the RNA is only modified by reducing the Ucontent in the coding region of the RNA by substituting U containingcodons by other codons encoding the same amino acids but comprisingfewer and preferably no U nucleosides the above may relate to thenucleotide sequence of the coding region only.

As used herein, the term “G+C content” or “GC content” refers to theamount of nucleosides of a particular RNA molecule or RNA sequence thatare either guanosine (G) or cytidine (C) typically expressed as apercent. Where the sequence of particular RNA is known, the G+C contentcan be determined using the formula:

$\frac{G + C}{A + U + G + C} \times 100$

wherein G, C, A and U refer to the number of each residue in theparticular RNA molecule or RNA sequence, to provide a percent GCcontent. If the nucleotide sequence of the RNA is only modified byreducing the U content in the coding region of the RNA by substituting Ucontaining codons by other codons encoding the same amino acids butcomprising fewer and preferably no U nucleosides the above may relate tothe nucleotide sequence of the coding region only.

There are a variety of different methods that can be used to substitutenucleosides and, in particular codons. For example, base substitutionscan be made in the DNA template used for making an RNA by standardsite-directed mutagenesis (See, for example, Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989 or 1991 edition). Alternatively, anentire RNA can be synthesized from DNA template enzymatically usingstandard in vitro transcription techniques. In the case of enzymaticallysynthesized RNAs it may be desirable to make other modifications, e.g.modifications to enhance RNA stability. For example, a cap can be addedto the synthetized RNA post-transcriptionally using capping enzymes orduring transcription. Likewise, a poly A tail can be addedpost-transcriptionally using enzymes, e.g., with poly A polymerase orduring transcription from the DNA template.

It should be understood that in addition to the sequence changesdescribed above, other sequence changes can be made in the RNA, e.g. thesubject RNA can be made more nuclease resistant by removing nucleasesensitive motifs. Certain RNAs are naturally unstable in a cell, andthis is normally due to the existence of destabilizing sequence motifswithin such unstable RNAs that are recognized by nucleases. If suchsequences exist in a RNA, they can be eliminated, replaced or modifiedby standard genetic engineering.

The term “nucleoside” relates to compounds which can be thought of asnucleotides without a phosphate group. While a nucleoside is anucleobase linked to a sugar (e.g. ribose or deoxyribose), a nucleotideis composed of a nucleoside and one or more phosphate groups. Examplesof nucleosides include cytidine, uridine, adenosine, and guanosine.

Uridine is a glycosylated pyrimidine-analog containing uracil attachedto a ribose ring (or more specifically, a ribofuranose) via aβ-N1-glycosidic bond. It is one of the five standard nucleosides whichmake up nucleic acids, the others being adenosine, thymidine, cytidineand guanosine. The five nucleosides are commonly abbreviated to theirone letter codes U, A, T, C and G, respectively. However, thymidine ismore commonly written as “dT” (“d” represents “deoxy”) as it contains a2′-deoxyribofuranose moiety rather than the ribofuranose ring found inuridine. This is because thymidine is found in deoxyribonucleic acid(DNA) and not ribonucleic acid (RNA). Conversely, uridine is found inRNA and not DNA. The remaining three nucleosides may be found in bothRNA and DNA. In RNA, they would be represented as A, C and G, whereas inDNA they would represented as dA, dC and dG.

According to the invention, a nucleic acid or nucleic acid moleculerefers to a nucleic acid which is preferably deoxyribonucleic acid (DNA)or ribonucleic acid (RNA). According to the invention, nucleic acidscomprise genomic DNA, cDNA, mRNA, recombinantly prepared and chemicallysynthesized molecules. According to the invention, a nucleic acid may bein the form of a single-stranded or double-stranded and linear orcovalently closed circular molecule. The term “nucleic acid” accordingto the invention also comprises a chemical derivatization of a nucleicacid on a nucleotide base, on the sugar or on the phosphate, and nucleicacids containing non-natural nucleotides and nucleotide analogs.

In the context of the present invention, the term “RNA” relates to amolecule which comprises ribonucleotide residues and preferably beingentirely or substantially composed of ribonucleotide residues. The term“ribonucleotide” relates to a nucleotide with a hydroxyl group at the2′-position of a β-D-ribofuranosyl group. The term “RNA” comprisesdouble-stranded RNA, single stranded RNA, isolated RNA such as partiallyor completely purified RNA, essentially pure RNA, synthetic RNA, andrecombinantly generated RNA such as modified RNA which differs fromnaturally occurring RNA by addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of a RNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in RNA molecules can also comprise non-standard nucleotides,such as non-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides. These altered RNAs can be referred toas analogs, particularly analogs of naturally-occurring RNAs. Accordingto the invention, RNA includes and preferably relates to mRNA. RNA suchas mRNA described herein may have a length of between about 500 to about10000, 5000 or 2000 nucleotides. In another embodiment, the RNA has alength of between about 500 to about 1000 nucleotides. In anotherembodiment, the RNA is greater than 30 nucleotides in length. In anotherembodiment, the RNA is greater than 50 nucleotides in length. In anotherembodiment, the length is at least 60 nucleotides. In anotherembodiment, the length is at least 80 nucleotides. In anotherembodiment, the length is at least 100 nucleotides. In anotherembodiment, the length is at least 120 nucleotides. In anotherembodiment, the length is at least 140 nucleotides. In anotherembodiment, the length is at least 160 nucleotides. In anotherembodiment, the length is at least 180 nucleotides. In anotherembodiment, the length is at least 200 nucleotides. In anotherembodiment, the length is at least 250 nucleotides. In anotherembodiment, the length is at least 300 nucleotides. In anotherembodiment, the length is at least 350 nucleotides. In anotherembodiment, the length is at least 400 nucleotides. In anotherembodiment, the length is at least 450 nucleotides. In anotherembodiment, the length is at least 500 nucleotides. In anotherembodiment, the length is at least 600 nucleotides. In anotherembodiment, the length is at least 700 nucleotides. In anotherembodiment, the length is at least 800 nucleotides. In anotherembodiment, the length is at least 900 nucleotides. In anotherembodiment, the length is at least 1000 nucleotides.

As used herein, the term “RNA” includes chemically modified,non-naturally occurring RNA. Such chemical modifications may render theRNA molecule more resistant to nucleases than a naturally occurring RNAmolecule. Exemplary modifications to a nucleic acid sequence of an RNAmolecule include, for example, the modification of a base, e.g., thechemical modification of a base. The term “chemical modification” asused herein, includes modifications which introduce chemistries whichdiffer from those seen in naturally occurring RNA. For example, chemicalmodifications include covalent modifications such as the introduction ofmodified nucleotides, e.g., nucleotide analogs, or the inclusion ofpendant groups which are not naturally found in RNA molecules. Suchmodifications include, but are not limited to pseudouridine,1-methylpseudouridine,1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine,2′-O-methylpseudouridine, 5-methyldihydrouridine, 5-methylcytidine,5-methyluridine, N6-methyladenosine, 2-thiouridine, 2′-O-methyluridine,1-methyladenosine, 2-methyladenosine, 2′-O-methyladenosine,2-methylthio-N6-methyladenosine, inosine, 1-methylinosine,3-methylcytidine, 2′-O-methylcytidine, 2-thiocytidine,N4-acetylcytidine, 5-formylcytidine, 5,2′-O-dimethylcytidine,1-methylguanosine, N2-methylguanosine, 7-methylguanosine,2′-O-methylguanosine, N2,N2-dimethylguanosine, dihydrouridine,5,2′-O-dimethyluridine, 4-thiouridine, 5-methyl-2-thiouridine,2-thio-2′-O-methyluridine, 5-hydroxyuridine, 5-methoxyuridine and3-methyluridine.

The term “mRNA” means “messenger-RNA” and relates to a transcript whichis generated by using a DNA template and encodes a peptide or protein.Typically, mRNA comprises a 5′ UTR, a protein coding region, a 3′ UTR,and a poly(A) sequence and may also comprise a 5′ cap. Several regionsof the mRNA molecule are not translated into protein including the 5′cap, 5′ UTR, 3′ UTR, and the poly(A) sequence.

The term “untranslated region” (or UTR) refers to either of twosections, one on each side of a coding region on a strand of mRNA. If itis found on the 5′ side, it is called the 5′ UTR, or if it is found onthe 3′ side, it is called the 3′ UTR.

The term “5′ untranslated region” relates to a region which is locatedat the 5′ end of a gene, upstream from the initiation codon of aprotein-encoding region, and which is transcribed but is not translatedinto an amino acid sequence, or to the corresponding region in an RNAmolecule. This region is important for the regulation of translation ofa transcript by differing mechanisms in viruses, prokaryotes andeukaryotes. While called untranslated, the 5′ UTR or a portion of it issometimes translated into a protein product. This product can thenregulate the translation of the main coding sequence of the mRNA. Inmany other organisms, however, the 5′ UTR is completely untranslated,instead forming complex secondary structure to regulate translation. The5′ UTR begins at the transcription start site and ends one nucleotide(nt) before the initiation sequence (usually AUG) of the coding region.In prokaryotes, the length of the 5′ UTR tends to be 3-10 nucleotideslong while in eukaryotes it tends to be anywhere from 100 to severalthousand nucleotides long. The elements of a eukaryotic and prokaryotic5′ UTR differ greatly. The prokaryotic 5′ UTR contains a ribosomebinding site (RBS), also known as the Shine Dalgarno sequence (AGGAGGU)which is usually 3-10 nucleotides upstream from the initiation codon.Meanwhile the eukaryotic 5′ UTR contains the Kozak consensus sequence(ACCAUGG), which contains the initiation codon.

The term “3′ untranslated region” relates to a region which is locatedat the 3′ end of a gene, downstream of the termination codon of aprotein-encoding region, and which is transcribed but is not translatedinto an amino acid sequence, or to the corresponding region in an RNAmolecule. Regulatory regions within the 3′ untranslated region caninfluence polyadenylation, translation efficiency, localization, andstability of the mRNA. The 3′ UTR contains both binding sites forregulatory proteins as well as microRNAs (miRNAs). The 3′ untranslatedregion typically extends from the termination codon for a translationproduct to the poly(A) sequence which is usually attached after thetranscription process. The 3′ untranslated regions of mammalian mRNAtypically have a homology region known as the AAUAAA hexanucleotidesequence. This sequence is presumably the poly(A) attachment signal andis frequently located from 10 to 30 bases upstream of the poly(A)attachment site.

According to the invention, a first polynucleotide region is consideredto be located downstream of a second polynucleotide region, if the 5′end of said first polynucleotide region is the part of said firstpolynucleotide region closest to the 3′ end of said secondpolynucleotide region.

Polyadenylation is the addition of a poly(A) sequence or tail to aprimary transcript RNA. The poly(A) sequence consists of multipleadenosine monophosphates residues called adenylates. In other words, itis a stretch of RNA that has only adenine bases. In eukaryotes,polyadenylation is part of the process that produces mature messengerRNA (mRNA) for translation. It, therefore, forms part of the largerprocess of gene expression. The process of polyadenylation begins as thetranscription of a gene finishes, or terminates. The 3′ most segment ofthe newly made pre-mRNA is first cleaved off by a set of proteins; theseproteins then synthesize the poly(A) sequence at the RNA's 3′ end. Thepoly(A) sequence is important for the nuclear export, translation, andstability of mRNA. The sequence is shortened over time, and, when it isshort enough, the mRNA is enzymatically degraded.

The terms “polyadenyl sequence”, “poly(A) sequence” or “poly(A) tail”refer to a sequence of adenylate residues which is typically located atthe 3′ end of an RNA molecule. The invention provides for such asequence to be attached during RNA transcription by way of a DNAtemplate on the basis of repeated thymidylate residues in the strandcomplementary to the coding strand, whereas said sequence is normallynot encoded in the DNA but is attached to the free 3′ end of the RNA bya template-independent RNA polymerase after transcription in thenucleus. According to the invention, in one embodiment, a poly(A)sequence has at least 20, preferably at least 40, preferably at least80, preferably at least 100 and preferably up to 500, preferably up to400, preferably up to 300, preferably up to 200, and in particular up to150 A nucleotides, preferably consecutive A nucleotides, and inparticular about 120 A nucleotides. The term “A nucleotides” or “A”refers to adenylate residues.

The term “5′ cap” refers to a cap structure found on the 5′ end of anmRNA molecule and generally consists of a guanosine nucleotide connectedto the mRNA via an unusual 5′ to 5′ triphosphate linkage. In oneembodiment, this guanosine is methylated at the 7-position. The term“conventional 5′ cap” refers to a naturally occurring RNA 5′ cap,preferably to the 7-methylguanosine cap (m7G). In the context of thepresent invention, the term “5′ cap” includes a 5′ cap analog thatresembles the RNA cap structure and is modified to possess the abilityto stabilize RNA if attached thereto, preferably in vivo and/or in acell. Providing an RNA with a 5′ cap or 5′ cap analog may be achieved byin vitro transcription of a DNA template in the presence of said 5′ capor 5′ cap analog, wherein said 5′ cap is co-transcriptionallyincorporated into the generated RNA strand, or the RNA may be generated,for example, by in vitro transcription, and the 5′ cap may be generatedpost-transcriptionally using capping enzymes, for example, cappingenzymes of vaccinia virus.

In one embodiment of the present invention, RNA is self-replicating RNA,such as single stranded self-replicating RNA. In one embodiment, theself-replicating RNA is single stranded RNA of positive sense. In oneembodiment, the self-replicating RNA is viral RNA or RNA derived fromviral RNA. In one embodiment, the self-replicating RNA is alphaviralgenomic RNA or is derived from alphaviral genomic RNA. In oneembodiment, the self-replicating RNA is a viral gene expression vector.In one embodiment, the virus is Semliki forest virus. In one embodiment,the self-replicating RNA contains one or more transgenes. In oneembodiment, if the RNA is viral RNA or derived from viral RNA, thetransgenes may partially or completely replace viral sequences such asviral sequences encoding structural proteins. In one embodiment, theself-replicating RNA is in vitro transcribed RNA.

In particular embodiments, the RNA according to the invention comprisesa population of different RNA molecules, e.g. a mixture of different RNAmolecules optionally encoding different peptides and/or proteins,whole-cell RNA, an RNA library, or a portion of thereof, e.g. a libraryof RNA molecules expressed in a particular cell type, such asundifferentiated cells, in particular stem cells such as embryonic stemcells, or a fraction of the library of RNA molecules such as RNA withenriched expression in undifferentiated cells, in particular stem cellssuch as embryonic stem cells relative to differentiated cells. Thus,according to the invention, the term “RNA” may include a mixture of RNAmolecules, whole-cell RNA or a fraction thereof, which may be obtainedby a process comprising the isolation of RNA from cells and/or byrecombinant means, in particular by in vitro transcription.

According to the invention, the term “gene” refers to a particularnucleic acid sequence which is responsible for producing one or morecellular products and/or for achieving one or more intercellular orintracellular functions. More specifically, said term relates to a DNAsection which comprises a nucleic acid coding for a specific protein ora functional or structural RNA molecule.

RNA can be isolated from cells, can be made from a DNA template, or canbe chemically synthesized using methods known in the art. In preferredembodiment, RNA is synthesized in vitro from a DNA template. In oneparticularly preferred embodiment, RNA, in particular mRNA is generatedby in vitro transcription from a DNA template. The in vitrotranscription methodology is known to the skilled person. For example,there is a variety of in vitro transcription kits commerciallyavailable. In one particularly preferred embodiment, RNA is in vitrotranscribed RNA (IVT RNA).

Preferably the RNA described herein is eukaryotic, preferably mammalianin origin. In preferred embodiments, the RNA comprises characteristicsof eukaryotic mRNA, e.g., the presence of a 5′ cap, and/or the presenceof a poly(A) sequence.

In a preferred embodiment, a nucleic acid molecule according to theinvention is a vector. The term “vector” is used here in its mostgeneral meaning and comprises any intermediate vehicles for a nucleicacid which, for example, enable said nucleic acid to be introduced intoprokaryotic and/or eukaryotic host cells and, where appropriate, to beintegrated into a genome. Such vectors are preferably replicated and/orexpressed in the cell. Vectors comprise plasmids, phagemids or virusgenomes. The term “plasmid”, as used herein, generally relates to aconstruct of extrachromosomal genetic material, usually a circular DNAduplex, which can replicate independently of chromosomal DNA.

The nucleic acids described herein may be recombinant and/or isolatedmolecules.

An “isolated molecule” as used herein, is intended to refer to amolecule which has been separated from its natural environment andpreferably is substantially free of other molecules such as othercellular material. The term “isolated nucleic acid” means according tothe invention that the nucleic acid has been (i) amplified in vitro, forexample by polymerase chain reaction (PCR), (ii) recombinantly producedby cloning, (iii) purified, for example by cleavage andgel-electrophoretic fractionation, or (iv) synthesized, for example bychemical synthesis. An isolated nucleic acid is a nucleic acid availableto manipulation by recombinant DNA techniques.

The term “recombinant” in the context of the present invention means“made through genetic engineering”. Preferably, a “recombinant object”such as a recombinant nucleic acid in the context of the presentinvention is not occurring naturally.

The term “naturally occurring” as used herein refers to the fact that anobject can be found in nature. For example, a peptide, protein ornucleic acid that is present in an organism (including viruses) and canbe isolated from a source in nature and which has not been intentionallymodified by man in the laboratory is naturally occurring.

As a nucleic acid, in particular RNA, for expression of more than onepeptide or protein, either of a nucleic acid type in which the differentpeptides or proteins are encoded in different nucleic acid molecules ora nucleic acid type in which the peptides or proteins are encoded in thesame nucleic acid molecule can be used.

The term “stability” of RNA relates to the “half-life” of RNA.“Half-life” relates to the period of time which is needed to eliminatehalf of the activity, amount, or number of molecules. In the context ofthe present invention, the half-life of an RNA is indicative for thestability of said RNA. The half-life of RNA may influence the “durationof expression” of the RNA. It can be expected that RNA having a longhalf-life will be expressed for an extended time period.

The term “expression” is used according to the invention in its mostgeneral meaning and comprises the production of RNA and/or peptides orproteins, e.g. by transcription and/or translation. With respect to RNA,the term “expression” or “translation” relates in particular to theproduction of peptides or proteins. It also comprises partial expressionof nucleic acids. Moreover, expression can be transient or stable.

According to the invention, terms such as “RNA expression”, “expressingRNA”, or “expression of RNA” relate to the production of peptide orprotein encoded by the RNA. Preferably, such terms relate to thetranslation of RNA so as to express, i.e. produce, peptide or proteinencoded by the RNA.

In the context of the present invention, the term “transcription”relates to a process, wherein the genetic code in a DNA sequence istranscribed into RNA. Subsequently, the RNA may be translated intoprotein. According to the present invention, the term “transcription”comprises “in vitro transcription”, wherein the term “in vitrotranscription” relates to a process wherein RNA, in particular mRNA, isin vitro synthesized in a cell-free system, preferably using appropriatecell extracts. Preferably, cloning vectors are applied for thegeneration of transcripts. These cloning vectors are generallydesignated as transcription vectors and are according to the presentinvention encompassed by the term “vector”. According to the presentinvention, RNA preferably is in vitro transcribed RNA (IVT-RNA) and maybe obtained by in vitro transcription of an appropriate DNA template.The promoter for controlling transcription can be any promoter for anyRNA polymerase. Particular examples of RNA polymerases are the T7, T3,and SP6 RNA polymerases. Preferably, the in vitro transcriptionaccording to the invention is controlled by a T7 or SP6 promoter. A DNAtemplate for in vitro transcription may be obtained by cloning of anucleic acid, in particular cDNA, and introducing it into an appropriatevector for in vitro transcription. The cDNA may be obtained by reversetranscription of RNA.

The term “translation” according to the invention relates to the processin the ribosomes of a cell by which a strand of messenger RNA directsthe assembly of a sequence of amino acids to make a peptide or protein.

The term “expression control sequence” comprises according to theinvention promoters, ribosome-binding sequences and other controlelements which control transcription of a gene or translation of thederived RNA. In particular embodiments of the invention, the expressioncontrol sequences can be regulated. The precise structure of expressioncontrol sequences may vary depending on the species or cell type butusually includes 5′ untranscribed and 5′ and 3′ untranslated sequencesinvolved in initiating transcription and translation, respectively, suchas TATA box, capping sequence, CAAT sequence and the like. Morespecifically, 5′ untranscribed expression control sequences include apromoter region which encompasses a promoter sequence for transcriptioncontrol of the functionally linked gene. Expression control sequencesmay also include enhancer sequences or upstream activator sequences.

The nucleic acid sequences specified herein, in particular transcribableand coding nucleic acid sequences, may be combined with any expressioncontrol sequences, in particular promoters, which may be homologous orheterologous to said nucleic acid sequences, with the term “homologous”referring to the fact that a nucleic acid sequence is also functionallylinked naturally to the expression control sequence, and the term“heterologous” referring to the fact that a nucleic acid sequence is notnaturally functionally linked to the expression control sequence.

The term “promoter” or “promoter region” refers to a DNA sequenceupstream (5′) of the coding sequence of a gene, which controlsexpression of said coding sequence by providing a recognition andbinding site for RNA polymerase. The promoter region may include furtherrecognition or binding sites for further factors involved in regulatingtranscription of said gene. A promoter may control transcription of aprokaryotic or eukaryotic gene. A promoter may be “inducible” andinitiate transcription in response to an inducer, or may be“constitutive” if transcription is not controlled by an inducer. Aninducible promoter is expressed only to a very small extent or not atall, if an inducer is absent. In the presence of the inducer, the geneis “switched on” or the level of transcription is increased. This isusually mediated by binding of a specific transcription factor.

Examples of promoters preferred according to the invention are promotersfor SP6, T3 or T7 polymerase.

Decreased immunogenicity of RNA according to the invention may result inenhanced expression of said RNA.

Terms such as “enhancement of expression”, “enhanced expression” or“increased expression” mean in the context of the present invention thatthe amount of peptide or protein expressed by a given number of RNAmolecules is higher than the amount of peptide or protein expressed bythe same number of RNA molecules, wherein expression of the RNAmolecules is performed under the same conditions except the conditionwhich results in the enhanced or increased expression of the RNA. Inthis context, “same conditions”, for example, refer to a situationwherein RNA sequences encoding the same peptide or protein areadministered to a subject by the same means and the amount of peptide orprotein is measured by the same means. The amount of peptide or proteinmay be given in moles, or by weight, e.g. in grams, or by mass or bypolypeptide activity, e.g. if the peptide or protein is an enzyme it maybe given as catalytic activity or if the peptide or protein is anantibody or antigen or a receptor it may be given as binding affinity.In one embodiment, terms such as “enhancement of expression”, “enhancedexpression” or “increased expression” mean in the context of the presentinvention that the amount of peptide or protein expressed by a givennumber of RNA molecules and within a given period of time is higher thanthe amount of peptide or protein expressed by the same number of RNAmolecules and within the same period of time. For example, the maximumvalue of peptide or protein expressed by a given number of RNA moleculesat a particular time point may be higher than the maximum value ofpeptide or protein expressed by the same number of RNA molecules. Inother embodiments, the maximum value of peptide or protein expressed bya given number of RNA molecules does not need to be higher than themaximum value of peptide or protein expressed by the same number of RNAmolecules, however, the average amount of peptide or protein expressedby the given number of RNA molecules within a given period of time maybe higher than the average amount of peptide or protein expressed by thesame number of RNA molecules. The latter cases are referred to herein as“higher level of expression” or “increased level of expression” andrelate to higher maximum values of expression and/or higher averagevalues of expression. Alternatively or additionally, terms such as“enhancement of expression”, “enhanced expression” or “increasedexpression” mean in the context of the present invention also that thetime in which peptide or protein is expressed by RNA molecules may belonger. Thus, in one embodiment, terms such as “enhancement ofexpression”, “enhanced expression” or “increased expression” mean in thecontext of the present invention also that the amount of peptide orprotein expressed by a given number of RNA molecules is higher than theamount of peptide or protein expressed by the same number of RNAmolecules since the period of time in which the RNA is stably presentand expressed is longer than the period of time in which the same numberof RNA molecules is stably present and expressed. These cases arereferred to herein also as “increased duration of expression”.Preferably, such longer time periods refer to expression for at least 48h, preferably for at least 72 h, more preferably for at least 96 h, inparticular for at least 120 h or even longer following administration ofRNA or following the first administration (e.g. in case of repeatedadministrations) of RNA.

The level of expression and/or duration of expression of RNA may bedetermined by measuring the amount, such as the total amount expressedand/or the amount expressed in a given time period, and/or the time ofexpression of the peptide or protein encoded by the RNA, for example, byusing an ELISA procedure, an immunohistochemistry procedure, aquantitative image analysis procedure, a Western Blot, massspectrometry, a quantitative immunohistochemistry procedure, or anenzymatic assay.

Preferably, according to the invention, following administration of RNAto a subject, the RNA is to be taken up by cells of the subject, i.e.cells of the subject are to be transfected with the RNA, for expressionof the peptide or protein encoded by the RNA.

The term “transfection” relates to the introduction of nucleic acids, inparticular RNA, into a cell. For purposes of the present invention, theterm “transfection” also includes the introduction of a nucleic acidinto a cell or the uptake of a nucleic acid by such cell, wherein thecell may be present in a subject, e.g., a patient. Thus, according tothe present invention, a cell for transfection of a nucleic acid can bepresent in vitro or in vivo, e.g. the cell can form part of an organ, atissue and/or an organism of a patient. According to the invention,transfection can be transient or stable. For some applications oftransfection, it is sufficient if the transfected genetic material isonly transiently expressed. Since the nucleic acid introduced in thetransfection process is usually not integrated into the nuclear genome,the foreign nucleic acid will be diluted through mitosis or degraded.Cells allowing episomal amplification of nucleic acids greatly reducethe rate of dilution. If it is desired that the transfected nucleic acidactually remains in the genome of the cell and its daughter cells, astable transfection must occur. RNA can be transfected into cells totransiently express its coded peptide or protein.

According to the present invention, any technique useful forintroducing, i.e. transferring or transfecting, nucleic acids into cellsin vitro may be used. Preferably, nucleic acid is transfected into cellsby standard techniques. Such techniques include electroporation,lipofection and microinjection. In one particularly preferred embodimentof the present invention, nucleic acid is introduced into cells byelectroporation. Electroporation or electropermeabilization relates to asignificant increase in the electrical conductivity and permeability ofthe cell plasma membrane caused by an externally applied electricalfield. It is usually used in molecular biology as a way of introducingsome substance into a cell.

According to the invention it is preferred that introduction of nucleicacid encoding a protein or peptide into cells or uptake of nucleic acidencoding a protein or peptide by cells results in expression of saidprotein or peptide. The cell may express the encoded peptide or proteinintracellularly (e.g. in the cytoplasm and/or in the nucleus), maysecrete the encoded peptide or protein, or may express it on thesurface.

According to the present invention, the administration of a nucleicacid, in particular RNA, is either achieved as naked nucleic acid or incombination with an administration reagent. Preferably, administrationof nucleic acids is in the form of naked nucleic acids. Preferably, theRNA is administered in combination with stabilizing substances such asRNase inhibitors. The present invention also envisions the repeatedintroduction of nucleic acids into cells to allow sustained expressionfor extended time periods. RNA can be administered with any carrierswith which RNA can be associated, e.g. by forming complexes with the RNAor forming vesicles in which the RNA is enclosed or encapsulated,resulting in increased stability of the RNA compared to naked RNA.Carriers useful according to the invention include, for example,lipid-containing carriers such as cationic lipids, liposomes, inparticular cationic liposomes, and micelles. Cationic lipids may formcomplexes with negatively charged nucleic acids. Any cationic lipid maybe used according to the invention.

According to the invention, nucleic acids may be directed to particularcells. In such embodiments, a carrier used for administering a nucleicacid to a cell (e.g. a retrovirus or a liposome) may have a boundtargeting molecule. For example, a molecule such as an antibody specificto a surface membrane protein on the target cell, or a ligand for areceptor on the target cell may be incorporated into or bound to thenucleic acid carrier. If administration of a nucleic acid by liposomesis desired, proteins binding to a surface membrane protein associatedwith endocytosis may be incorporated into the liposome formulation inorder to enable targeting and/or absorption. Such proteins includecapsid proteins or fragments thereof which are specific to a particularcell type, antibodies to proteins that are internalized, proteinstargeting an intracellular site, and the like.

Interferons are important cytokines characterized by antiviral,antiproliferative and immunomodulatory activities. Interferons areproteins that alter and regulate the transcription of genes within acell by binding to interferon receptors on the regulated cell's surface,thereby preventing viral replication within the cells. The interferonscan be grouped into two types. IFN-gamma is the sole type II interferon;all others are type I interferons. Type I and type II interferons differin gene structure (type II interferon genes have three exons; type I,one), chromosome location (in humans, type II is located onchromosome-12; the type I interferon genes are linked and onchromosome-9), and the types of tissues where they are produced (type Iinterferons are synthesized ubiquitously, type II by lymphocytes). TypeI interferons competitively inhibit each others binding to cellularreceptors, while type II interferon has a distinct receptor. Accordingto the invention, the term “interferon” or “IFN” preferably relates totype I interferons, in particular IFN-alpha and IFN-beta.

According to the invention, the term “host cell” refers to any cellwhich can be transformed or transfected with an exogenous nucleic acid.The term “host cell” comprises, according to the invention, prokaryotic(e.g. E. coli) or eukaryotic cells (e.g. yeast cells and insect cells).Particular preference is given to mammalian cells such as cells fromhumans, mice, hamsters, pigs, goats, primates. The cells may be derivedfrom a multiplicity of tissue types and comprise primary cells and celllines. Specific examples include keratinocytes, peripheral bloodleukocytes, bone marrow stem cells and embryonic stem cells. In otherembodiments, the host cell is an antigen-presenting cell, in particulara dendritic cell, a monocyte or a macrophage. A nucleic acid may bepresent in the host cell in a single or in several copies and, in oneembodiment is expressed in the host cell.

According to the present invention, the term “peptide” comprises oligo-and polypeptides and refers to substances which comprise two or more,preferably 3 or more, preferably 4 or more, preferably 6 or more,preferably 8 or more, preferably 10 or more, preferably 13 or more,preferably 16 or more, preferably 20 or more, and up to preferably 50,preferably 100 or preferably 150, consecutive amino acids linked to oneanother via peptide bonds. The term “protein” refers to large peptides,preferably peptides having at least 151 amino acids, but the terms“peptide” and “protein” are used herein usually as synonyms.

The terms “peptide” and “protein” comprise according to the inventionsubstances which contain not only amino acid components but alsonon-amino acid components such as sugars and phosphate structures, andalso comprise substances containing bonds such as ester, thioether ordisulfide bonds.

According to the present invention, a nucleic acid such as RNA mayencode a peptide or protein. Accordingly, a nucleic acid such as RNA maycontain a coding region (open reading frame (ORF)) encoding a peptide orprotein. Said nucleic may express the encoded peptide or protein. Forexample, said nucleic acid may be a nucleic acid encoding and expressingan antigen or a pharmaceutically active peptide or protein such as animmunologically active compound (which preferably is not an antigen). Inthis respect, an “open reading frame” or “ORF” is a continuous stretchof codons beginning with a start codon and ending with a stop codon.

According to the invention, the term “RNA encoding a peptide or protein”means that the RNA, if present in the appropriate environment,preferably within a cell, can direct the assembly of amino acids toproduce, i.e. express, the peptide or protein during the process oftranslation. Preferably, RNA according to the invention is able tointeract with the cellular translation machinery allowing translation ofthe peptide or protein.

According to the invention, in one embodiment, RNA comprises or consistsof pharmaceutically active RNA. A “pharmaceutically active RNA” may beRNA that encodes a pharmaceutically active peptide or protein.

The term “pharmaceutically active peptide or protein” includes a peptideor protein that can be used in the treatment of a subject where theexpression of a peptide or protein would be of benefit, e.g., inameliorating the symptoms of a disease or disorder. For example, apharmaceutically active protein can replace or augment proteinexpression in a cell which does not normally express a protein or whichmisexpresses a protein, e.g., a pharmaceutically active protein cancompensate for a mutation by supplying a desirable protein. In addition,a “pharmaceutically active peptide or protein” can produce a beneficialoutcome in a subject, e.g., can be used to produce a protein to whichvaccinates a subject against an infectious disease. Preferably, a“pharmaceutically active peptide or protein” has a positive oradvantageous effect on the condition or disease state of a subject whenadministered to the subject in a therapeutically effective amount.Preferably, a pharmaceutically active peptide or protein has curative orpalliative properties and may be administered to ameliorate, relieve,alleviate, reverse, delay onset of or lessen the severity of one or moresymptoms of a disease or disorder. A pharmaceutically active peptide orprotein may have prophylactic properties and may be used to delay theonset of a disease or to lessen the severity of such disease orpathological condition. The term “pharmaceutically active peptide orprotein” includes entire proteins or polypeptides, and can also refer topharmaceutically active fragments thereof. It can also includepharmaceutically active analogs of a peptide or protein. The term“pharmaceutically active peptide or protein” includes peptides andproteins that are antigens, i.e., the peptide or protein elicits animmune response in a subject which may be therapeutic or partially orfully protective.

“Effective amount” or “therapeutically effective amount” (with respectto e.g. RNA, peptide or protein) refers to an amount sufficient to exerta therapeutic effect. In another embodiment, the term refers to anamount of RNA sufficient to elicit expression of a detectable amount ofthe peptide or protein encoded by the RNA.

Examples of pharmaceutically active proteins include, but are notlimited to, cytokines and immune system proteins such as immunologicallyactive compounds (e.g., interleukins, colony stimulating factor (CSF),granulocyte colony stimulating factor (G-CSF), granulocyte-macrophagecolony stimulating factor (GM-CSF), erythropoietin, tumor necrosisfactor (TNF), interferons, integrins, addressins, seletins, homingreceptors, T cell receptors, immunoglobulins, soluble majorhistocompatibility complex antigens, immunologically active antigenssuch as bacterial, parasitic, or viral antigens, allergens,autoantigens, antibodies), hormones (insulin, thyroid hormone,catecholamines, gonadotrophines, trophic hormones, prolactin, oxytocin,dopamine, bovine somatotropin, leptins and the like), growth hormones(e.g., human grown hormone), growth factors (e.g., epidermal growthfactor, nerve growth factor, insulin-like growth factor and the like),growth factor receptors, enzymes (tissue plasminogen activator,streptokinase, cholesterol biosynthetic or degradative, steriodogenicenzymes, kinases, phosphodiesterases, methylases, de-methylases,dehydrogenases, cellulases, proteases, lipases, phospholipases,aromatases, cytochromes, adenylate or guanylaste cyclases, neuramidasesand the like), receptors (steroid hormone receptors, peptide receptors),binding proteins (growth hormone or growth factor binding proteins andthe like), transcription and translation factors, tumor growthsuppressing proteins (e.g., proteins which inhibit angiogenesis),structural proteins (such as collagen, fibroin, fibrinogen, elastin,tubulin, actin, and myosin), blood proteins (thrombin, serum albumin,Factor VII, Factor VIII, insulin, Factor IX, Factor X, tissueplasminogen activator, protein C, von Wilebrand factor, antithrombinIII, glucocerebrosidase, erythropoietin granulocyte colony stimulatingfactor (GCSF) or modified Factor VIII, anticoagulants and the like.

In one embodiment, the pharmaceutically active protein according to theinvention is a cytokine which is involved in regulating lymphoidhomeostasis, preferably a cytokine which is involved in and preferablyinduces or enhances development, priming, expansion, differentiationand/or survival of T cells. In one embodiment, the cytokine is aninterleukin. In one embodiment, the pharmaceutically active proteinaccording to the invention is an interleukin selected from the groupconsisting of IL-2, IL-7, IL-12, IL-15, and IL-21.

The term “immunologically active compound” relates to any compoundaltering an immune response, preferably by inducing and/or suppressingmaturation of immune cells, inducing and/or suppressing cytokinebiosynthesis, and/or altering humoral immunity by stimulating antibodyproduction by B cells. Immunologically active compounds possess potentimmunostimulating activity including, but not limited to, antiviral andantitumor activity, and can also down-regulate other aspects of theimmune response, for example shifting the immune response away from aTH2 immune response, which is useful for treating a wide range of TH2mediated diseases. Immunologically active compounds can be useful asvaccine adjuvants.

In one embodiment, RNA that codes for an antigen such adisease-associated antigen is administered to a mammal, in particular iftreating a mammal having a disease involving the antigen is desired. TheRNA is preferably taken up into the mammal's antigen-presenting cells(monocytes, macrophages, dendritic cells or other cells). An antigenictranslation product of the RNA is formed and the product is displayed onthe surface of the cells for recognition by T cells. In one embodiment,the antigen or a product produced by optional procession thereof isdisplayed on the cell surface in the context of MHC molecules forrecognition by T cells through their T cell receptor leading to theiractivation.

The present invention also includes “variants” of the peptides,proteins, or amino acid sequences described herein.

For the purposes of the present invention, “variants” of an amino acidsequence comprise amino acid insertion variants, amino acid additionvariants, amino acid deletion variants and/or amino acid substitutionvariants.

Amino acid insertion variants comprise insertions of single or two ormore amino acids in a particular amino acid sequence. In the case ofamino acid sequence variants having an insertion, one or more amino acidresidues are inserted into a particular site in an amino acid sequence,although random insertion with appropriate screening of the resultingproduct is also possible.

Amino acid addition variants comprise amino- and/or carboxy-terminalfusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50,or more amino acids.

Amino acid deletion variants are characterized by the removal of one ormore amino acids from the sequence, such as by removal of 1, 2, 3, 5,10, 20, 30, 50, or more amino acids. The deletions may be in anyposition of the protein. Amino acid deletion variants that comprise thedeletion at the N-terminal and/or C-terminal end of the protein are alsocalled N-terminal and/or C-terminal truncation variants.

Amino acid substitution variants are characterized by at least oneresidue in the sequence being removed and another residue being insertedin its place. Preference is given to the modifications being inpositions in the amino acid sequence which are not conserved betweenhomologous proteins or peptides and/or to replacing amino acids withother ones having similar properties. Preferably, amino acid changes inprotein variants are conservative amino acid changes, i.e.,substitutions of similarly charged or uncharged amino acids. Aconservative amino acid change involves substitution of one of a familyof amino acids which are related in their side chains. Naturallyoccurring amino acids are generally divided into four families: acidic(aspartate, glutamate), basic (lysine, arginine, histidine), non-polar(alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), and uncharged polar (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine) amino acids.Phenylalanine, tryptophan, and tyrosine are sometimes classified jointlyas aromatic amino acids.

Preferably the degree of similarity, preferably identity between a givenamino acid sequence and an amino acid sequence which is a variant ofsaid given amino acid sequence will be at least about 60%, 65%, 70%,80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity oridentity is given preferably for an amino acid region which is at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90% or about 100% of the entire length of thereference amino acid sequence. For example, if the reference amino acidsequence consists of 200 amino acids, the degree of similarity oridentity is given preferably for at least about 20, at least about 40,at least about 60, at least about 80, at least about 100, at least about120, at least about 140, at least about 160, at least about 180, orabout 200 amino acids, preferably continuous amino acids. In preferredembodiments, the degree of similarity or identity is given for theentire length of the reference amino acid sequence. The alignment fordetermining sequence similarity, preferably sequence identity can bedone with art known tools, preferably using the best sequence alignment,for example, using Align, using standard settings, preferablyEMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

“Sequence similarity” indicates the percentage of amino acids thateither are identical or that represent conservative amino acidsubstitutions. “Sequence identity” between two amino acid sequencesindicates the percentage of amino acids or nucleotides that areidentical between the sequences.

The term “percentage identity” is intended to denote a percentage ofamino acid residues which are identical between the two sequences to becompared, obtained after the best alignment, this percentage beingpurely statistical and the differences between the two sequences beingdistributed randomly and over their entire length. Sequence comparisonsbetween two amino acid sequences are conventionally carried out bycomparing these sequences after having aligned them optimally, saidcomparison being carried out by segment or by “window of comparison” inorder to identify and compare local regions of sequence similarity. Theoptimal alignment of the sequences for comparison may be produced,besides manually, by means of the local homology algorithm of Smith andWaterman, 1981, Ads App. Math. 2, 482, by means of the local homologyalgorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by meansof the similarity search method of Pearson and Lipman, 1988, Proc. NatlAcad. Sci. USA 85, 2444, or by means of computer programs which usethese algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA inWisconsin Genetics Software Package, Genetics Computer Group, 575Science Drive, Madison, Wis.).

The percentage identity is calculated by determining the number ofidentical positions between the two sequences being compared, dividingthis number by the number of positions compared and multiplying theresult obtained by 100 so as to obtain the percentage identity betweenthese two sequences.

Homologous amino acid sequences exhibit according to the invention atleast 40%, in particular at least 50%, at least 60%, at least 70%, atleast 80%, at least 90% and preferably at least 95%, at least 98 or atleast 99% identity of the amino acid residues.

According to the invention, a variant of a peptide or protein preferablyhas a functional property of the peptide or protein from which it hasbeen derived.

The term “disease” or “disorder” refers to an abnormal condition thataffects the body of an individual. A disease is often construed as amedical condition associated with specific symptoms and signs. A diseasemay be caused by factors originally from an external source, such asinfectious disease, or it may be caused by internal dysfunctions, suchas autoimmune diseases. As used herein the term “disease” or “disorder”includes, in particular, a condition which would benefit from theexpression of a peptide or protein (as described above), e.g, asdemonstrated by a reduction in and/or an amelioration of symptoms.

According to the invention, the term “disease” also refers to cancerdiseases. The terms “cancer disease” or “cancer” (medical term:malignant neoplasm) refer to a class of diseases in which a group ofcells display uncontrolled growth (division beyond the normal limits),invasion (intrusion on and destruction of adjacent tissues), andsometimes metastasis (spread to other locations in the body via lymph orblood). These three malignant properties of cancers differentiate themfrom benign tumors, which are self-limited, and do not invade ormetastasize. Most cancers form a tumor, i.e. a swelling or lesion formedby an abnormal growth of cells (called neoplastic cells or tumor cells),but some, like leukemia, do not. Examples of cancers include, but arenot limited to, carcinoma, lymphoma, blastoma, sarcoma, glioma andleukemia. More particularly, examples of such cancers include bonecancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skincancer, cancer of the head or neck, cutaneous or intraocular malignantmelanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of theanal region, stomach cancer, colon cancer, breast cancer, prostatecancer, uterine cancer, carcinoma of the sexual and reproductive organs,Hodgkin's disease, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the bladder, cancer of the kidney, renal cellcarcinoma, carcinoma of the renal pelvis, neoplasms of the centralnervous system (CNS), neuroectodermal cancer, spinal axis tumors,glioma, meningioma, and pituitary adenoma. The term “cancer” accordingto the invention also comprises cancer metastases.

The term “infectious disease” refers to any disease which can betransmitted from individual to individual or from organism to organism,and is caused by a microbial agent (e.g. common cold). Examples ofinfectious diseases include viral infectious diseases, such as AIDS(HIV), hepatitis A, B or C, herpes, herpes zoster (chicken-pox), Germanmeasles (rubella virus), yellow fever, dengue etc. flaviviruses,influenza viruses, hemorrhagic infectious diseases (Marburg or Ebolaviruses), and severe acute respiratory syndrome (SARS), bacterialinfectious diseases, such as Legionnaire's disease (Legionella),sexually transmitted diseases (e.g. chlamydia or gonorrhea), gastriculcer (Helicobacter), cholera (Vibrio), tuberculosis, diphtheria,infections by E. coli, Staphylococci, Salmonella or Streptococci(tetanus); infections by protozoan pathogens such as malaria, sleepingsickness, leishmaniasis; toxoplasmosis, i.e. infections by Plasmodium,Trypanosoma, Leishmania and Toxoplasma; or fungal infections, which arecaused e.g. by Cryptococcus neoformans, Histoplasma capsulatum,Coccidioides immitis, Blastomyces dermatitidis or Candida albicans.

The term “autoimmune disease” refers to any disease in which the bodyproduces an immunogenic (i.e. immune system) response to someconstituent of its own tissue. In other words, the immune system losesits ability to recognize some tissue or system within the body as selfand targets and attacks it as if it were foreign. Autoimmune diseasescan be classified into those in which predominantly one organ isaffected (e.g. hemolytic anemia and anti-immune thyroiditis), and thosein which the autoimmune disease process is diffused through many tissues(e.g. systemic lupus erythematosus). For example, multiple sclerosis isthought to be caused by T cells attacking the sheaths that surround thenerve fibers of the brain and spinal cord. This results in loss ofcoordination, weakness, and blurred vision. Autoimmune diseases areknown in the art and include, for instance, Hashimoto's thyroiditis,Grave's disease, lupus, multiple sclerosis, rheumatic arthritis,hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus,celiac disease, Crohn's disease, colitis, diabetes, scleroderma,psoriasis, and the like.

According to the invention, an immune response may be stimulated byintroducing into a subject a suitable mRNA which codes for an antigen ora fragment thereof, e.g., a disease-associated antigen.

The term “antigen” relates to an agent comprising an epitope againstwhich an immune response is to be generated. The term “antigen” includesin particular peptides and proteins. The term “antigen” also includesagents, which become antigenic—and sensitizing—only throughtransformation (e.g. intermediately in the molecule or by completionwith body protein). An antigen is preferably presentable by cells of theimmune system such as antigen presenting cells like dendritic cells ormacrophages. In addition, an antigen or a processing product thereof ispreferably recognizable by a T or B cell receptor, or by animmunoglobulin molecule such as an antibody. In a preferred embodiment,the antigen is a disease-associated antigen, such as a tumor-associatedantigen, a viral antigen, or a bacterial antigen.

The term “disease-associated antigen” is used in it broadest sense torefer to any antigen associated with a disease. A disease-associatedantigen is a molecule which contains epitopes that will stimulate ahost's immune system to make a cellular antigen-specific immune responseand/or a humoral antibody response against the disease. Thedisease-associated antigen may therefore be used for therapeuticpurposes. Disease-associated antigens are preferably associated withinfection by microbes, typically microbial antigens, or associated withcancer, typically tumors.

The term “disease involving an antigen” refers to any disease whichimplicates an antigen, e.g. a disease which is characterized by thepresence and/or expression of an antigen. The disease involving anantigen can be an infectious disease, an autoimmune disease, or a cancerdisease or simply cancer. As mentioned above, the antigen may be adisease-associated antigen, such as a tumor-associated antigen, a viralantigen, or a bacterial antigen.

In one embodiment, a disease-associated antigen is a tumor-associatedantigen. In this embodiment, the present invention may be useful intreating cancer or cancer metastasis. Preferably, the diseased organ ortissue is characterized by diseased cells such as cancer cellsexpressing a disease-associated antigen and/or being characterized byassociation of a disease-associated antigen with their surface.Immunization with intact or substantially intact tumor-associatedantigens or fragments thereof such as MHC class I and class II peptidesor nucleic acids, in particular mRNA, encoding such antigen or fragmentmakes it possible to elicit a MHC class I and/or a class II typeresponse and, thus, stimulate T cells such as CD8+ cytotoxic Tlymphocytes which are capable of lysing cancer cells and/or CD4+ Tcells. Such immunization may also elicit a humoral immune response (Bcell response) resulting in the production of antibodies against thetumor-associated antigen. In one embodiment, the term “tumor-associatedantigen” refers to a constituent of cancer cells which may be derivedfrom the cytoplasm, the cell surface and the cell nucleus. Inparticular, it refers to those antigens which are produced, preferablyin large quantity, intracellularly or as surface antigens on tumorcells. Examples for tumor-associated antigens include HER2, EGFR, VEGF,CAMPATH1-antigen, CD22, CA-125, HLA-DR, Hodgkin-lymphoma or mucin-1, butare not limited thereto.

According to the present invention, a tumor-associated antigenpreferably comprises any antigen which is characteristic for tumors orcancers as well as for tumor or cancer cells with respect to type and/orexpression level. In one embodiment, the term “tumor-associated antigen”relates to proteins that are under normal conditions, i.e. in a healthysubject, specifically expressed in a limited number of organs and/ortissues or in specific developmental stages, for example, thetumor-associated antigen may be under normal conditions specificallyexpressed in stomach tissue, preferably in the gastric mucosa, inreproductive organs, e.g., in testis, in trophoblastic tissue, e.g., inplacenta, or in germ line cells, and are expressed or aberrantlyexpressed in one or more tumor or cancer tissues. In this context, “alimited number” preferably means not more than 3, more preferably notmore than 2 or 1. The tumor-associated antigens in the context of thepresent invention include, for example, differentiation antigens,preferably cell type specific differentiation antigens, i.e., proteinsthat are under normal conditions specifically expressed in a certaincell type at a certain differentiation stage, cancer/testis antigens,i.e., proteins that are under normal conditions specifically expressedin testis and sometimes in placenta, and germ line specific antigens. Inthe context of the present invention, the tumor-associated antigen ispreferably not or only rarely expressed in normal tissues or is mutatedin tumor cells. Preferably, the tumor-associated antigen or the aberrantexpression of the tumor-associated antigen identifies cancer cells. Inthe context of the present invention, the tumor-associated antigen thatis expressed by a cancer cell in a subject, e.g., a patient sufferingfrom a cancer disease, is preferably a self-protein in said subject. Inpreferred embodiments, the tumor-associated antigen in the context ofthe present invention is expressed under normal conditions specificallyin a tissue or organ that is non-essential, i.e., tissues or organswhich when damaged by the immune system do not lead to death of thesubject, or in organs or structures of the body which are not or onlyhardly accessible by the immune system. Preferably, a tumor-associatedantigen is presented in the context of MHC molecules by a cancer cell inwhich it is expressed.

Examples for differentiation antigens which ideally fulfill the criteriafor tumor-associated antigens as contemplated by the present inventionas target structures in tumor immunotherapy, in particular, in tumorvaccination are the cell surface proteins of the Claudin family, such asCLDN6 and CLDN18.2. These differentiation antigens are expressed intumors of various origins, and are particularly suited as targetstructures in connection with antibody-mediated cancer immunotherapy dueto their selective expression (no expression in a toxicity relevantnormal tissue) and localization to the plasma membrane.

Further examples for antigens that may be useful in the presentinvention are p53, ART-4, BAGE, beta-catenin/m, Bcr-abL CAMEL, CAP-1,CASP-8, CDCl₂7/m, CDK4/m, CEA, CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M,ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2/neu, HPV-E7, HPV-E6,HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1/Melan-A, MC1R,Myosin/m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190minor BCR-abL, Pm1/RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 orRU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN,TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2, TPTE and WT, preferably WT-1.

The term “viral antigen” refers to any viral component having antigenicproperties, i.e. being able to provoke an immune response in anindividual. The viral antigen may be a viral ribonucleoprotein or anenvelope protein.

The term “bacterial antigen” refers to any bacterial component havingantigenic properties, i.e. being able to provoke an immune response inan individual. The bacterial antigen may be derived from the cell wallor cytoplasm membrane of the bacterium.

“Antigen processing” refers to the degradation of an antigen intoprocession products, which are fragments of said antigen (e.g., thedegradation of a protein into peptides) and the association of one ormore of these fragments (e.g., via binding) with MEW molecules forpresentation by cells, preferably antigen presenting cells to specific Tcells.

The term “immune response”, as used herein, relates to a reaction of theimmune system such as to immunogenic organisms, such as bacteria orviruses, cells or substances. The term “immune response” includes theinnate immune response and the adaptive immune response. Preferably, theimmune response is related to an activation of immune cells, aninduction of cytokine biosynthesis and/or antibody production. It ispreferred that the immune response comprises the steps of activation ofantigen presenting cells, such as dendritic cells and/or macrophages,presentation of an antigen or fragment thereof by said antigenpresenting cells and activation of cytotoxic T cells due to thispresentation.

The term “treat” or “treatment” relates to any treatment which improvesthe health status and/or prolongs (increases) the lifespan of anindividual. Said treatment may eliminate the disease in an individual,arrest or slow the development of a disease in an individual, inhibit orslow the development of a disease in an individual, decrease thefrequency or severity of symptoms in an individual, and/or decrease therecurrence in an individual who currently has or who previously has hada disease.

In particular, the term “treatment of a disease” includes curing,shortening the duration, ameliorating, slowing down or inhibitingprogression or worsening of a disease or the symptoms thereof.

The term “immunotherapy” relates to a treatment preferably involving aspecific immune reaction and/or immune effector function(s).

The term “immunization” or “vaccination” describes the process oftreating a subject for therapeutic or prophylactic reasons.

The term “in vivo” relates to the situation in a subject.

The terms “subject” and “individual” are used interchangeably and relateto mammals. For example, mammals in the context of the present inventionare humans, non-human primates, domesticated animals such as dogs, cats,sheep, cattle, goats, pigs, horses etc., laboratory animals such asmice, rats, rabbits, guinea pigs, etc. as well as animals in captivitysuch as animals of zoos. The term “animal” as used herein also includeshumans. The term “subject” may also include a patient, i.e., an animal,preferably a human having a disease.

The nucleic acids such as RNA described herein, in particular when usedfor the treatments described herein, may be present in the form of apharmaceutical composition or kit comprising the nucleic acid andoptionally one or more pharmaceutically acceptable carriers, diluentsand/or excipients.

Pharmaceutical compositions are preferably sterile and contain aneffective amount of the nucleic acid.

Pharmaceutical compositions are usually provided in a uniform dosageform and may be prepared in a manner known in the art. Thepharmaceutical composition may, e.g., be in the form of a solution orsuspension.

The pharmaceutical composition may comprise salts, buffer substances,preservatives, carriers, diluents and/or excipients all of which arepreferably pharmaceutically acceptable. The term “pharmaceuticallyacceptable” refers to the non-toxicity of a material which does notinterfere with the action of the active component(s) of thepharmaceutical composition.

Salts which are not pharmaceutically acceptable may be used forpreparing pharmaceutically acceptable salts and are included in theinvention. Pharmaceutically acceptable salts of this kind comprise, in anon-limiting way, those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic acids, and the like. Pharmaceuticallyacceptable salts may also be prepared as alkali metal salts or alkalineearth metal salts, such as sodium salts, potassium salts or calciumsalts.

Suitable buffer substances for use in the pharmaceutical compositioninclude acetic acid in a salt, citric acid in a salt, boric acid in asalt and phosphoric acid in a salt.

Suitable preservatives for use in the pharmaceutical composition includebenzalkonium chloride, chlorobutanol, paraben and thimerosal.

The term “carrier” refers to an organic or inorganic component, of anatural or non-natural (synthetic) nature, with which the activecomponent is combined in order to facilitate, enhance or enableapplication. According to the invention, the term “carrier” alsoincludes one or more compatible solid or liquid fillers, diluents orencapsulating substances, which are suitable for administration to apatient.

Possible carrier substances for parenteral administration are, e.g.,sterile water, glucose solutions, Ringer, Ringer lactate, sterile sodiumchloride solution, polyalkylene glycols, hydrogenated naphthalenes and,in particular, biocompatible lactide polymers, lactide/glycolidecopolymers or polyoxyethylene/polyoxy-propylene copolymers.

The term “excipient” when used herein is intended to indicate allsubstances which may be present in a pharmaceutical composition andwhich are not active ingredients such as, e.g., carriers, binders,lubricants, thickeners, surface active agents, preservatives,emulsifiers, buffers, flavoring agents, or colorants.

The pharmaceutical compositions described herein may be administered viaany conventional route, such as by parenteral administration includingby injection or infusion. Administration is preferably parenterally,e.g. intravenously, intraarterially, subcutaneously, in the lymph node,intradermally or intramuscularly.

Compositions suitable for parenteral administration usually comprise asterile aqueous or non-aqueous preparation of the active compound, whichis preferably isotonic to the blood of the recipient. Examples ofcompatible carriers and solvents are Ringer's solution and isotonicsodium chloride solution. In addition, usually sterile, fixed oils areused as solution or suspension medium.

The agents and compositions described herein are preferably administeredin effective amounts. An “effective amount” refers to the amount whichachieves a desired reaction or a desired effect alone or together withfurther doses. In the case of treatment of a particular disease or of aparticular condition, the desired reaction preferably relates toinhibition of the course of the disease. This comprises slowing down theprogress of the disease and, in particular, interrupting or reversingthe progress of the disease. The desired reaction in a treatment of adisease or of a condition may also be delay of the onset or a preventionof the onset of said disease or said condition.

An effective amount of an agent or composition described herein willdepend on the condition to be treated, the severeness of the disease,the individual parameters of the patient, including age, physiologicalcondition, size and weight, the duration of treatment, the type of anaccompanying therapy (if present), the specific route of administrationand similar factors. Accordingly, the doses administered of the agentsdescribed herein may depend on several of these parameters. In the casethat a reaction in a patient is insufficient with an initial dose,higher doses (or effectively higher doses achieved by a different, morelocalized route of administration) may be used.

The present invention is described in detail by the following figuresand examples which should be construed by way of illustration only andnot by way of limitation. On the basis of the description and theexamples, further embodiments are accessible to the skilled worker andare likewise within the scope of the invention.

EXAMPLES Example 1: Generation and Purification of mRNAs

DNA encoding mouse EPO or canine EPO was ordered from and synthesized byGenScript. Coding mRNAs were produced using T7 RNA polymerase and onlythe 4 basic nucleotides ATP, GTP, UTP and CTP (Megascript, Ambion) fromlinearized plasmids encoding RNAs encoding murine erythropoietin (EPO).All mRNA contained identical 5′UTR corresponding to tobacco etch viral(TEV) leader (Gallie D R, Tanguay R L, Leathers V.: (1995) The tobaccoetch viral 5′ leader and poly(A) tail are functionally synergisticregulators of translation. Gene 165, 233-238) and identical 3′UTRs.Additionally, all IVT mRNA contained 100 nt-long polyA-tail interruptedby a linker (GCAUAUGACU) at nt 30 downstream from the 3′ UTR. All IVTmRNA was capped using the m7G capping enzyme and 2′-O-methyltransferase(CellScript, Madison, Wis.).

Inter alia, the following murine EPO mRNAs were generated: mEPO:wild-type (wt) in which the coding sequence of EPO contained 55% GC and125 uridine; 2) omEPO: GC-rich with 63% GC and 92 uridine; and 3) A-richEPO with 53% GC and 80 uridine. The produced mRNAs were HPLC purified asdescribed (Karikó, K, Muramatsu, H, Ludwig, J and Weissman, D (2011).Generating the optimal mRNA for therapy: HPLC purification eliminatesimmune activation and improves translation of nucleoside-modified,protein-encoding mRNA. Nucleic Acids Res 39:e142). The nucleotidecomposition of the non-coding region can be modified as well. Forpurpose of demonstrating the effect of the invention only the codingregion was modified as described. All other elements of the mRNAs werekept constant as described above.

Example 2: Preparation of Lipid Nanoparticles (LNP) Entrapping mRNA

LNPs were prepared by using a microfluidic mixing device, theNanoAssemblr Benchtop Instrument (Precision NanoSystems, Vancouver, BC).One volume of lipids mixture in ethanol (DLin-KC2-DMA (DLin-KC2-DMA wassynthetized according to Semple, S C, Akinc, A, Chen, J, Sandhu, A P,Mui, B L, Cho, C K et al. (2010) and Rational design of cationic lipidsfor siRNA delivery. Nat Biotechnol 28: 172-176), Cholesterol (SigmaAldrich, Taufkirchen Germany), DSPC(1,2-Distearoyl-sn-glycero-3-phosphocholine, Corden Pharm, LiestalSwitzerland), mPEG2000-CeramideC16 (Avanti Polar Lipids, Alabster, Ala.,USA) at an appropriate molar ratio) and 3 volumes of RNA in citratebuffer 100 mM, pH 5.4 (17:1 w/w lipid/RNA) were mixed through themicrofluidic cartridge at a combined flow rate of 12 mL/min (3 mL/minfor ethanol and 9 mL/min for aqueous buffer). The resultant phase wasdirectly mixed with 2 volumes of citrate buffer 100 mM, pH 5,4. Themixture was then dialyzed against phosphate buffered saline (PBS) for2.5 h to remove ethanol in a Slide-A-Lyser dialysis cassette (10K MWCO,ThermoFisher Scientific). The particles were then re-concentrated byultrafiltration using Amicon Ultra Centrifugal filters (30 kDa NMWL,Merck Millipore) to a total RNA concentration of about 0.3 to 0.5 mg/mL.siRNA encapsulation efficiency was determined by the Quant-iT RiboGreenRNA assay (Life Technologies). Briefly, the encapsulation efficiency wasdetermined using the RNA binding dye RiboGreen by comparing fluorescencebetween LNPs in the presence and absence of 0.5% Triton X-100. In theabsence of detergent, fluorescence can be measured from accessible freeRNA only, whereas in the presence of detergent, fluorescence is measuredfrom the total RNA amount.

Example 3: Preparation of TransIT-Complexed mRNA

mRNA generated according to example 1 was complexed to TransIT-mRNA(Minis Bio, Madison, Wis.) according to the manufacturer. In a regularpolypropylene tube, first Dulbecco's modified Eagle's medium (DMEM) wasmeasured then 1 μg mRNA was added quickly, followed by 1.1 μlTransIT-mRNA reagent and 0.7 μl Boost reagent to obtain the complex in afinal volume of 100 μl DMEM. The components were combined and vortexedfor 20 second, then let stand for 2 min and injected immediately. Forcomplexing different amounts of mRNA the volumes of the reagents and thefinal volume were scaled proportionally.

Example 4: Preparation of Liposomal mRNA Formulation

Liposomes were manufactured by a modification of the so-called ethanolinjection technique, where an ethanolic solution of the lipids isinjected under stirring into an aqueous phase. As lipids, the syntheticcationic lipid DOTMA and the phospholipid DOPE in a molar ratio of 2:1were used. A manufacturing protocol resulting in liposomes in the sizerange of about 400 nm, in order to obtain the appropriate size of theRNA-lipoplexes was used. Briefly, an ethanol solution of the lipids wasprepared, sterile filtrated, and injected into water for injection (WFI)under stirring to obtain a lipid concentration in the aqueous phase ofabout 6 to 10 mM. Thus a liposome raw dispersion with pre-defined sizewas obtained. Subsequently the liposome raw dispersion was filtrated inorder to reduce the amount of larger aggregates. Subsequently, the lipidcontent of the liposome dispersion was determined, and, depending on theresult, the liposomes were diluted with WFI to a final concentration ofabout 4 mM. The liposomes were filled in depyrogenated and sterilized 10mL glass vials which were closed with sterilized stoppers and flip-offcrimping caps.

The mRNA was prepared to obtain liposomal 20 μg mRNA in a 200 μl finalvolume. First, the RNA was diluted to obtain 1 μg RNA/μl of HEPES/EDTAin a final concentration of 10 mM HEPES/0.1 mM EDTA. An aliquot of 20 μlof RNA was measured into an eppendorf tube then 146 μl water and 20 μlof 1.5 M NaCl were added and mixed well. The RNA solution was incubatedat room temperature for 2 minutes, then 14 μl of liposomal solution wasadded, vortexed, then incubated at room temperature for 10 min. Finallythe liposomal mRNA was used for the experiments or injected.

Example 5: Administration/Injection of Formulated mRNAs

Formulated mRNAs according to examples 2 to 4 were administered in vitroto human dendritic cells or in vivo to BALB/c mice as follows:

Human dendritic cells were transfected with 0.1 μg of TransIT-complexedmRNA according to example 3. The purification of DCs from PBMCs used themethod originally described by Sallusto and Lanzavecchia (Sallusto, F.,and A. Lanzavecchia. 1994. Efficient presentation of soluble antigen bycultured human dendritic cells is maintained by granulocyte/macrophagecolony-stimulating factor plus interleukin 4 and downregulated by tumornecrosis factor a. J. Exp. Med. 179:1109) with minor modification.Briefly, monocytes were purified from PBMC by Ficoll density gradientcentrifugation. Human CD14+ cells were selected by positive selectionusing CD14 MicroBeads (Miltenyi Biotec). To generate immature DC,purified monocytes were cultured for 4 days in RPMI 1640 supplementedwith glutamine (2 mM), HEPES (15 mM), 1% NHS (Sigma), GM-CSF (50 ng/ml)and IL-4 (100 ng/ml). Cells were seeded into 96-well plate at 1×105cells/200 μl/well density in culture medium supplemented with 10% FCS.Cells were transfected by adding 17 μl of TransIT-complexed 0.1 μgmRNA/well. The complex was generated as described in Example 3. Cellswere cultured overnight and the medium was harvested at 24 hposttransfection. Murine EPO level was measured by ELISA (ErythropoietinDuoSet ELISA Development kit, R&D) and murine IFN was measured also byELISA (eBioscience, Platinum ELISA)

20 μg of liposomal-complexed mRNAs according to example 4 were injectedintravenously, by retro-orbital route to 6-weeks old BALB/c mice, 5animals/group. At 6 h following injection animals were bleed and EDTAwas used to inhibit coagulation of the drawn blood. Plasma was separatedby centrifugation and collected. Murine EPO level was measured by ELISA(Erythropoietin DuoSet ELISA Development kit, R&D) and murine IFN wasmeasured also by ELISA (eBioscience, Platinum ELISA)

10 μg of LNP-formulated mRNAs according to example 2 were injectedintravenously, by retro-orbital route to 6-weeks old BALB/c mice, 5animals/group. At 6 h following injection, animals were bleed and EDTAwas used to inhibit coagulation of the drawn blood. Plasma was separatedby centrifugation and collected. Murine EPO level was measured by ELISA(Erythropoietin DuoSet ELISA Development kit, R&D) and murine IFN wasmeasured also by ELISA (eBioscience, Platinum ELISA). Results are shownin FIG. 2.

SEQUENCES Amino Acid Sequence:

All used/codon-optimized murine EPO nucleic acid sequences encode thesame murine EPO protein with the following amino acid sequence:

MGVPERPTLLLLLSLLLIPLGLPVLCAPPRLICDSRVLERYILEAKEAENVTMGCAEGPRLSENITVPDTKVNFYAWKRMEVEEQAIEVWQGLSLLSEAILQAQALLANSSQPPETLQLHIDKAISGLRSLTSLLRVLGAQKELMSPPDTTPPAPLRTLTVDTFCKLFRVYANFLRGKLKLYTGEVCRRGDR.

Nucleic Acid Sequences:

(1) Original murine EPO coding sequences, also called murine EPO (mEPO)or wild-type EPO (wt EPO):

ATGGGGGTGCCCGAACGTCCCACCCTGCTGCTTTTACTCTCCTTGCTACTGATTCCTCTGGGCCTCCCAGTCCTCTGTGCTCCCCCACGCCTCATCTGCGACAGTCGAGTTCTGGAGAGGTACATCTTAGAGGCCAAGGAGGCAGAAAATGTCACGATGGGTTGTGCAGAAGGTCCCAGACTGAGTGAAAATATTACAGTCCCAGATACCAAAGTCAACTTCTATGCTTGGAAAAGAATGGAGGTGGAAGAACAGGCCATAGAAGTTTGGCAAGGCCTGTCCCTGCTCTCAGAAGCCATCCTGCAGGCCCAGGCCCTGCTAGCCAATTCCTCCCAGCCACCAGAGACCCTTCAGCTTCATATAGACAAAGCCATCAGTGGTCTACGTAGCCTCACTTCACTGCTTCGGGTACTGGGAGCTCAGAAGGAATTGATGTCGCCTCCAGATACCACCCCACCTGCTCCACTCCGAACACTCACAGTGGATACTTTCTGCAAGCTCTTCCGGGTCTACGCCAACTTCCTCCGGGGGAAACTGAAGCTGTACACGGGAGAGGTCTGCAGGAGAGGGGACAGGTGA

(2) GC-rich murine EPO coding sequence, also called optimized murine EPO(omEPO). This sequence was codon-optimized by GeneArt AG, Regensburg:

ATGGGCGTCCCCGAAAGGCCTACCCTGCTGCTGCTCCTGTCTCTGCTCCTGATCCCCCTGGGACTGCCCGTGCTGTGCGCCCCTCCCAGGCTGATCTGCGACAGCAGGGTGCTGGAAAGATACATCCTGGAAGCCAAAGAGGCCGAGAACGTCACAATGGGCTGCGCCGAGGGCCCCAGACTGAGCGAGAACATCACCGTGCCCGACACCAAGGTCAACTTCTACGCCTGGAAGAGGATGGAAGTGGAGGAACAGGCCATCGAGGTCTGGCAGGGACTGTCTCTGCTGTCCGAGGCCATCCTGCAGGCCCAGGCTCTGCTGGCCAATTCTAGCCAGCCCCCCGAGACACTGCAGCTGCACATCGACAAGGCCATCAGCGGCCTGAGAAGCCTGACCAGCCTGCTGAGGGTGCTGGGAGCCCAGAAAGAGCTGATGAGCCCCCCTGACACCACCCCCCCTGCCCCCCTGAGGACCCTGACCGTGGACACCTTCTGCAAGCTGTTCAGGGTGTACGCCAACTTCCTGAGGGGCAAGCTGAAGCTGTACACCGGCGAGGTCTGCAGACGGGGCGACAGATGA

(3) A-rich murine EPO coding sequence:

ATGGGAGTGCCAGAAAGACCAACCCTGCTGCTGCTGCTCAGCCTGCTACTGATCCCACTGGGACTCCCAGTCCTCTGCGCACCACCAAGACTCATCTGCGACAGCAGAGTGCTGGAAAGATACATCCTAGAAGCAAAAGAAGCAGAAAACGTCACGATGGGATGCGCAGAAGGACCAAGACTGAGCGAAAACATCACAGTCCCAGACACCAAAGTCAACTTCTACGCATGGAAAAGAATGGAAGTGGAAGAACAGGCAATAGAAGTGTGGCAAGGACTGAGCCTGCTCAGCGAAGCAATCCTGCAGGCACAGGCACTGCTAGCAAACAGCAGCCAGCCACCAGAAACCCTGCAGCTGCACATAGACAAAGCAATCAGCGGACTAAGAAGCCTCACCAGCCTGCTGAGAGTACTGGGAGCACAGAAAGAACTGATGAGCCCACCAGACACCACCCCACCAGCACCACTCAGAACACTCACAGTGGACACTTTCTGCAAACTCTTCAGAGTCTACGCAAACTTCCTCAGAGGAAAACTGAAACTGTACACGGGAGAAGTCTGCAGAAGAGGGGACAGATGA

(4) Super optimized murine EPO (somEPO), optimized by Entelechon usingEntelechon's proprietary algorithm and codon-optimized human EPO (Kim, CH, Oh, Y and Lee, T H (1997). Codon optimization for high-levelexpression of human erythropoietin (EPO) in mammalian cells. Gene 199:293-301). This sequence was used previously, e.g. described in Karikó,K. et al. Kariko K, Muramatsu H, Keller J M, Weissman D (2012) Increasederythropoiesis in mice injected with submicrogram quantities ofpseudouridine-containing mRNA encoding erythropoietin. Mol Ther20:948-953:

ATGGGAGTTCCTGAAAGACCAACTCTGTTGCTCTTGCTGTCTTTGCTGCTGATTCCTCTGGGTCTTCCGGTGCTTTGCGCACCTCCCAGGCTTATCTGCGATAGCAGGGTGCTTGAGAGGTACATCCTGGAAGCTAAAGAAGCCGAAAACGTGACCATGGGCTGCGCCGAGGGCCCTAGGCTCAGTGAAAACATTACTGTTCCCGATACGAAAGTCAATTTCTACGCCTGGAAGCGGATGGAAGTGGAGGAACAGGCCATAGAGGTGTGGCAAGGTCTGTCTCTCCTGAGCGAGGCAATCCTTCAAGCCCAGGCTCTGCTGGCCAATTCAAGCCAGCCACCCGAGACCCTCCAGCTGCACATTGACAAGGCTATCAGCGGTCTGAGATCCCTGACGTCCCTGTTGCGAGTCCTGGGCGCTCAGAAGGAGCTGATGAGTCCACCCGATACCACACCTCCAGCACCGCTCCGCACACTCACTGTGGACACCTTTTGTAAACTGTTCAGAGTCTACGCCAACTTTCTGCGAGGCAAGCTGAAGCTCTATACAGGAGAGGTGTGTAGGAGAGGAGACCGGTGA

(5) GC-maximized murine EPO (as described in Thess, A., Grund, S., Mui,B. L., Hope, M. J., Baumhof, P., Fotin-Mleczek, M., and Schlake, T.(2015) Sequence-engineered mRNA without chemical nucleosidemodifications enables an effective protein therapy in large animals(Molecular Therapy 23, 1457-1465):

ATGGGCGTGCCCGAGCGGCCGACCCTGCTCCTGCTGCTCAGCCTGCTGCTCATCCCCCTGGGGCTGCCCGTCCTCTGCGCCCCCCCGCGCCTGATCTGCGACTCCCGGGTGCTGGAGCGCTACATCCTCGAGGCCAAGGAGGCGGAGAACGTGACCATGGGCTGCGCCGAGGGGCCCCGGCTGAGCGAGAACATCACGGTCCCCGACACCAAGGTGAACTTCTACGCCTGGAAGCGCATGGAGGTGGAGGAGCAGGCCATCGAGGTCTGGCAGGGCCTGTCCCTCCTGAGCGAGGCCATCCTGCAGGCGCAGGCCCTCCTGGCCAACTCCAGCCAGCCCCCGGAGACACTGCAGCTCCACATCGACAAGGCCATCTCCGGGCTGCGGAGCCTGACCTCCCTCCTGCGCGTGCTGGGCGCGCAGAAGGAGCTCATGAGCCCGCCCGACACGACCCCCCCGGCCCCGCTGCGGACCCTGACCGTGGACACGTTCTGCAAGCTCTTCCGCGTCTACGCCAACTTCCTGCGGGGCAAGCTGAAGCTCTACACCGGGGAGGTGTGCCGCCGGGGCGACCGCTGA

(6) Amino Acid Sequence:

All used/codon-optimized canine EPO (Canis lupus familiaris) nucleicacid sequences encode the same canine EPO protein with the followingamino acid sequence (Swiss-Prot No. J9NYY7):

MCEPAPPKPTQSAWHSFPECPALLLLLSLLLLPLGLPVLGAPPRLICDSRVLERYILEAREAENVTMGCAQGCSFSENITVPDTKVNFYTWKRMDVGQQALEVWQGLALLSEAILRGQALLANASQPSETPQLHVDKAVSSLRSLTSLLRALGAQKEAMSLPEEASPAPLRTFTVDTLCKLFRIYSNFLRGKLTLYTGEA CRRGDR

Nucleic Acid Sequences:

(7) Original canine EPO coding sequences, also called canine EPO (cEPO)or wild-type EPO (wt EPO):

ATGTGCGAACCCGCCCCACCTAAGCCCACTCAGTCTGCTTGGCACAGTTTCCCCGAATGTCCAGCTCTCCTGCTGCTGCTCTCCCTGCTGCTCCTGCCCCTCGGGCTGCCTGTGCTGGGCGCTCCTCCAAGACTCATCTGCGACAGCAGGGTGCTGGAGCGGTACATCCTGGAGGCTAGAGAAGCCGAGAATGTCACCATGGGGTGTGCTCAGGGATGCTCCTTCAGCGAGAACATCACCGTGCCCGACACTAAGGTGAACTTCTATACATGGAAGCGGATGGATGTGGGACAGCAGGCCCTCGAAGTGTGGCAGGGCCTCGCTCTGCTGTCTGAAGCCATCCTGAGGGGACAGGCCCTCCTGGCTAATGCCAGCCAGCCTTCAGAGACCCCCCAGCTGCACGTGGACAAAGCCGTGTCAAGCCTGAGATCCCTCACAAGCCTCCTGAGGGCTCTGGGCGCTCAGAAGGAAGCCATGTCTCTGCCAGAGGAAGCCAGCCCTGCCCCACTCAGGACCTTCACTGTCGATACCCTGTGCAAGCTGTTCAGGATCTATTCCAACTTTCTGAGGGGCAAACTGACACTCTATACTGGGGAGGCT TGTAGGCGGGGAGACCGATGA

(8) A-rich canine EPO coding sequence:

ATGTGCGAACCAGCACCACCTAAACCAACACAGAGCGCATGGCACAGCTTCCCAGAATGCCCAGCACTGCTGCTGCTGCTCAGCCTGCTACTGCTGCCACTGGGACTCCCAGTCCTCGGAGCACCACCAAGACTCATCTGCGACAGCAGAGTGCTGGAAAGATACATCCTAGAAGCAAGAGAAGCAGAAAACGTCACGATGGGATGCGCACAAGGATGCAGCTTCAGCGAAAACATCACAGTCCCAGACACCAAAGTCAACTTCTACACATGGAAAAGAATGGACGTGGGACAGCAGGCACTGGAAGTGTGGCAAGGACTGGCACTGCTCAGCGAAGCAATCCTGAGAGGACAGGCACTGCTAGCAAACGCAAGCCAGCCAAGCGAAACCCCACAGCTGCACGTAGACAAAGCAGTGAGCAGCCTAAGAAGCCTCACCAGCCTGCTGAGAGCACTGGGAGCACAGAAAGAAGCCATGAGCCTGCCAGAAGAAGCCAGCCCAGCACCACTCAGAACATTCACAGTGGACACCCTGTGCAAACTGTTCAGAATATACAGCAACTTCCTCAGAGGAAAACTGACACTGTACACGGGAGAAGCT TGCAGAGGAGGAGACAGATGA

We claim:
 1. A method of decreasing immunogenicity of RNA, said methodcomprising modifying the nucleotide sequence of the RNA by reducing theuridine (U) content, wherein said reduction of the U content comprisesan elimination of U nucleosides from the nucleotide sequence of the RNAand/or a substitution of U nucleosides by nucleosides other than U inthe nucleotide sequence of the RNA.
 2. The method of claim 1 comprisingthe steps of: providing the nucleotide sequence of a first RNA,designing the nucleotide sequence of a second RNA, said nucleotidesequence of the second RNA comprising a reduced U content compared tothe nucleotide sequence of the first RNA, and, optionally, providing thesecond RNA.
 3. A method of providing a nucleic acid molecule for RNAtranscription comprising the steps of: providing a first DNA sequenceencoding the nucleotide sequence of a first RNA, designing a second DNAsequence encoding the nucleotide sequence of a second RNA, saidnucleotide sequence of the second RNA comprising a reduced U contentcompared to the nucleotide sequence of the first RNA, wherein saidreduction of the U content comprises an elimination of U nucleosidesfrom the nucleotide sequence of the RNA and/or a substitution of Unucleosides by nucleosides other than U in the nucleotide sequence ofthe RNA, and providing a nucleic acid molecule comprising the second DNAsequence.
 4. The method of any one of claims 1-3, wherein the RNAencodes at least one peptide or protein.
 5. The method of claim 4,wherein the peptide or protein is pharmaceutically active or antigenic.6. The method of claim 4 or 5, wherein the amino acid sequence of thepeptide or protein encoded by the RNA modified by reducing the U contentis identical to the amino acid sequence of the peptide or proteinencoded by the non-modified RNA.
 7. The method of any one of claims 1-6,wherein said reduced U content renders the RNA modified by reducing theU content less immunogenic compared to the non-modified RNA.
 8. Themethod of any one of claims 1-7, wherein the U content in the RNAmodified by reducing the U content is reduced by at least 10%,preferably at least 20%, more preferably at least 30% compared to thenon-modified RNA.
 9. The method of any one of claims 1-8, wherein the Ucontent is reduced in one or more of the 5′ untranslated region, thecoding region and the 3′ untranslated region of the RNA.
 10. The methodof any one of claims 1-9, wherein the U content is reduced in the codingregion of the RNA.
 11. The method of any one of claims 1-10, whereinsaid reduction of the U content comprises a substitution of Unucleosides by nucleosides other than U in the nucleotide sequence ofthe RNA.
 12. The method of any one of claims 1-11, wherein saidnucleosides other than U are selected from the group consisting ofadenosine (A), guanosine (G), 5-methyluridine (m5U) and cytidine (C).13. The method of any one of claims 1-12, wherein said reduction of theU content comprises a substitution of U nucleosides by adenosine (A)nucleosides.
 14. The method of any one of claims 1-13, wherein saidreduction of the U content comprises altering codons which comprise atleast one U nucleoside by other codons that encode the same amino acidsbut comprise fewer U nucleosides and preferably comprise no Unucleosides.
 15. The method of any one of claims 1-14, furthercomprising introducing at least one analogue of a naturally occurringnucleoside into the nucleotide sequence of the RNA.
 16. The method ofclaim 15, wherein introducing the analogue of a naturally occurringnucleoside into the nucleotide sequence of the RNA reducesimmunogenicity of the RNA.
 17. The method of claim 15 or 16, whereinintroducing at least one analogue of a naturally occurring nucleosideinto the nucleotide sequence of the RNA comprises a substitution of Unucleosides by pseudouridines.
 18. The method of any one of claims 1-17,wherein the RNA is mRNA.
 19. A method of obtaining RNA comprising thesteps of (i) providing a nucleic acid molecule for RNA transcriptionaccording to the method of any one of claims 3-18, and (ii) transcribingRNA using the nucleic acid molecule as a template.
 20. A modified RNAhaving decreased immunogenicity compared to naturally occurring RNA,said modified RNA having a nucleotide sequence comprising a reduced Ucontent compared to said naturally occurring RNA, wherein said reductionof the U content comprises an elimination of U nucleosides from thenucleotide sequence of the RNA and/or a substitution of U nucleosides bynucleosides other than U in the nucleotide sequence of the RNA.
 21. Amethod of treating a subject using RNA comprising the steps of (i)decreasing immunogenicity of RNA according to the method of any one ofclaims 1, 2, and 4-18, and (ii) administering the RNA to the subject.22. A method of treating a subject using RNA comprising the steps of (i)obtaining RNA according to the method of claim 19, and (ii)administering the RNA to the subject.
 23. A method of treating a subjectcomprising administering the RNA of claim 20 to the subject.